Translational control system using RNA-protein interaction motif

ABSTRACT

A translational control method using an RNA-protein interaction motif is provided. The method comprises a step of introducing an mRNA having: a 5′UTR regulation structure comprising: (1) a cap structure at the 5′ terminus, (2) a spacer positioned on the 3′ side of the cap structure, and (3) one or more RNA motifs positioned on the 3′ side of the spacer, which comprises an RNA-protein interaction motif-derived nucleotide sequence or a variant thereof; and a nucleotide sequence encoding a target protein gene on the 3′ side of the 5′UTR regulation structure, into a cell in the presence of a protein specifically binding to the RNA motifs, wherein a translational level is decreased as the number of bases of the spacer decreases, and the translational level is decreased as the number of the RNA motifs increases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.14/415,009, which is the U.S. National Stage application ofPCT/JP2013/069958, filed Jul. 16, 2013, which claims priority from U.S.provisional application 61/672,219, filed Jul. 16, 2012.

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-WEB and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 19, 2018, isnamed sequence.txt and is 34 KB.

TECHNICAL FIELD

The present invention relates to a translational control system using anRNA-protein interaction motif.

BACKGROUND ART

Introduction of multiple genes into cells and translation and expressionof them are increasingly required for engineering and understandingbiological systems. Small-molecule-responsive translational regulatorysystems are widely known and have been used for expressing transgenes.Such a method is a technique to equally regulate expression of multiplegenes.

Up to the present, quantitative regulation of the expression of aprotein from an exogenous gene in a eukaryote largely depends on atranscription factor responsive to added small molecules such astetracycline (Deans T L, Cantor C R, Collins J J (2007) A tunablegenetic switch based on RNAi and repressor proteins for regulating geneexpression in mammalian cells. Cell 130:363-372). The activity of thetranscription factor is determined depending upon the concentration ofadded effector molecules. Such a combination of effector molecules and atranscription factor equally regulates transcription levels of multipletarget genes in a cell. FIG. 16 is a conceptual diagram of such aconventional system.

Post-transcriptional regulation of a target gene in a mammalian cell hasbeen also reported (Okano H et al. (2005) Function of RNA-bindingprotein Musashi-1 in stem cells. Exp Cell Res. 306: 349-356).

The present inventors designed a target mRNA containing a kink-turn RNAmotif, that is, a binding RNA motif of an archaeal ribosomal protein,L7Ae protein, and reported a “translation OFF switch” system forstrongly decreasing the translation of this mRNA (WO2009/066757). FIG.15 illustrates the structure of the kink-turn RNA motif and a briefoutline of the system. The system functions as an On-Off switch.However, the use is restricted for quantitative translationalregulation.

SUMMARY OF INVENTION Technical Problem

It is extremely significant to individually and independently regulateexpression of multiple exogenous genes by using a single regulatoryfactor. Such a system has, however, not yet been reported. The presentinvention was accomplished for solving this problem. The presentinventors have conceived translational regulation for an mRNA encoding atarget gene performed by using an RNA motif of an RNA-proteincomplex-derived nucleotide sequence or a variant thereof and a proteinspecifically binding to the RNA motif. As a result, the presentinventors have found that quantitative translational repression can beperformed by inserting the RNA motif in an mRNA 5′-untranslated region(hereinafter abbreviated as 5′UTR) while altering the number of insertedRNA motifs and a distance of the insertion portion from the 5′ terminus.Thus, the present invention was accomplished.

Solution to Problem

Specifically, according to one embodiment, the present inventionprovides a translational control method using an RNA-protein interactionmotif, comprising a step of introducing an mRNA having: a 5′UTRregulation structure comprising (1) a cap structure at the 5′ terminus,(2) a spacer positioned on the 3′ side of the cap structure, and (3) oneor more RNA motifs positioned on the 3′ side of the spacer, whichcomprises an RNA-protein interaction motif-derived nucleotide sequenceor a variant thereof; and a nucleotide sequence encoding a targetprotein gene on the 3′ side of the 5′UTR regulation structure, into acell in the presence of a protein specifically binding to the RNAmotifs, wherein a translational level is decreased as the number ofbases of the spacer decreases, and the translational level is decreasedas the number of the RNA motifs increases.

According to another embodiment, the present invention provides an mRNAtranslational level decreasing method, comprising the step of providing,on the 5′ side of a nucleotide sequence encoding a target protein gene,a 5′UTR regulation structure comprising

(1) a cap structure at the 5′ terminus,

(2) a spacer positioned on the 3′ side of the cap structure, and

(3) one or more RNA motifs positioned on the 3′ side of the spacer,which comprises an RNA-protein interaction motif-derived nucleotidesequence or a variant thereof, wherein translational level is decreasedas the number of bases of the spacer decreases, and the translationallevel is decreased as the number of the RNA motifs increases.

According to still another embodiment, the present invention provides anmRNA comprising: a 5′UTR regulation structure comprising (1) a capstructure at the 5′ terminus, (2) a spacer positioned on the 3′ side ofthe cap structure, and (3) one or more RNA motifs positioned on the 3′side of the spacer, which comprises an RNA-protein interactionmotif-derived nucleotide sequence or a variant thereof; and a nucleotidesequence encoding a target protein gene on the 3′ side of the 5′UTRregulation structure, wherein translational level of the target proteinis decreased in the presence of a protein specifically binding to theRNA motifs.

According to still another embodiment, the present invention provides amethod for selecting an exogenous mRNA that translates a protein at afreely selected level in a cell, comprising the steps of (1) introducingthe mRNA into a cell that expresses a protein specifically binding to acorresponding RNA motif; and (2) measuring a translational level of theprotein to identify the mRNA providing a desired translational level.

According to still another embodiment, the present invention provides amethod for regulating expression levels of target proteins from aplurality of different mRNAs encoding different target protein genesindependently at different levels, comprising the steps of introducing afirst mRNA, which has a cap structure at the 5′ terminus, contains anucleotide sequence encoding a first target protein gene, and has, onthe 3′ side of the cap structure and the 5′ side of an initiation codon,a first regulation structure comprising a spacer and one or more firstRNA motifs of an RNA-protein interaction motif-derived nucleotidesequence or a variant thereof, into a cell in the presence of a proteinspecifically binding to the first RNA motifs; and introducing a secondmRNA, which has a cap structure at the 5′ terminus, contains anucleotide sequence encoding a second target protein gene, and has, onthe 3′ side of the cap structure and the 5′ side of an initiation codon,a second regulation structure comprising a spacer, and one or moresecond RNA motifs of an RNA-protein interaction motif-derived nucleotidesequence or a variant thereof, into a cell in the presence of a proteinspecifically binding to the second RNA motifs, wherein the firstregulation structure and the second regulation structure are differentfrom each other in the number of bases of the spacer and/or the numberof the RNA motifs, and the first RNA motif and the second RNA motif arethe same as each other, or the first RNA motif and the second RNA motifare variants specifically binding to the same protein but havingdifferent dissociation constants for the same protein.

According to still another embodiment, the present invention provides atranslational regulation method using an RNA-protein interaction motif,comprising the step of introducing an mRNA, which has a 5′UTR regulationstructure comprising (1) a cap structure on the 5′ terminus, (2) one ormore RNA motifs positioned on the 3′ side of the cap structure, of anRNA-protein interaction motif-derived nucleotide sequence or a variantthereof, and (3) an ON switch cassette positioned on the 3′ side of theRNA motifs and having a sequence comprising (a) a bait open readingframe (a bait ORF), (b) intron and (c) an internal ribosome entry site(IRES); and a nucleotide sequence encoding a target protein gene on the3′ side of the 5′UTR regulation structure, into a cell, and startingtranslation of the target protein by a protein specifically binding tothe RNA motifs, wherein the bait ORF is a sequence comprising a stopcodon within 500 bases from the 3′ terminus thereof.

According to still another embodiment, the present invention provides anmRNA, comprising a 5′UTR regulation structure comprising (1) a capstructure at the 5′ terminus, (2) one or more RNA motifs positioned onthe 3′ side of the cap structure, of an RNA-protein interactionmotif-derived nucleotide sequence or a variant thereof, and (3) an ONswitch cassette positioned on the 3′ side of the RNA motifs and having asequence comprising (a) a bait open reading frame (a bait ORF), (b)intron and (c) an internal ribosome entry site (IRES); and a nucleotidesequence encoding a target protein gene on the 3′ side of the 5′UTRregulation structure site and having a nucleotide sequence encoding agene of a target protein, wherein the bait ORF is a sequence comprisinga stop codon in more than 320 bases from the intron.

Advantageous Effects of Invention

According to the present invention, expression levels of differentproteins from a plurality of different mRNAs can be controlledindependently at different levels in the presence of one and the sameactive factor, and thus, quantitative translational regulation can berealized.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a conceptual diagram illustrating variability of an mRNA 5′UTRregulation structure capable of performing translational control for atarget protein in accordance with the number of RNA motifs and the baselength of a spacer.

FIGS. 2(A) and 2(B) are schematic diagrams of an mRNA having a 5′UTRregulation structure comprising a spacer and one Kt motif.

FIG. 3 is a diagram illustrating an embodiment in which a spacer isfurther inserted in an mRNA having a 5′UTR regulation structurecomprising one Kt motif and encoding ECFP for tuning with the baselength of the spacer altered.

FIG. 4 is a graph illustrating relationships, obtained in an mRNA havinga 5′UTR regulation structure comprising a spacer and one Kt motif and anmRNA having a 5′UTR regulation structure comprising a spacer and one dKtmotif, between a distance of the corresponding motif from the 5′terminus and translational efficiency.

FIGS. 5(A) and 5(B) are diagrams illustrating secondary structures of aKl motif and a Kl2 motif, that is, variants of the Kt motif FIG. 5(A)illustrates the structure of the K-loop RNA motif (SEQ ID NO:2). FIG.5(B) illustrates the structure of the Kl2 motif (SEQ ID NO:3).

FIGS. 6(A), 6(B) and 6(C) are schematic diagrams of an mRNA having a5′UTR regulation structure comprising multiple Kl motifs.

FIG. 7 is a graph illustrating a relationship, obtained in an mRNAhaving a 5′UTR regulation structure comprising a spacer and a Kl motif,among the number of Kl motifs, the length of the spacer (i.e., adistance of the Kl motif from the 5′ terminus) and the translationalefficiency.

FIG. 8 is a graph illustrating relationships, obtained in an mRNA havinga 5′UTR regulation structure comprising a spacer and a Kl motif and anmRNA having a 5′UTR regulation structure comprising a spacer and a Ktmotif, among the number of motifs, the length of the spacer (i.e., adistance of the corresponding motif from the 5′ terminus) and thetranslational efficiency.

FIG. 9 is a conceptual diagram illustrating a system for simultaneouslyand independently regulating, in the presence of a single triggerprotein, translation of two mRNAs different from each other in thestructure of a 5′UTR regulation structure and a target protein toencode.

FIG. 10 is a diagram of relative mRNA levels, attained aftertranscription, of four mRNAs having different 5′UTR regulationstructures.

FIG. 11 is a diagram illustrating fluorescence profiles of EGFP and ECFPobtained by simultaneously expressing nine sets of mRNAs each set ofwhich is composed of two mRNAs different from each other in the 5′UTRregulation structure and the target protein gene.

FIGS. 12(A) and 12(B) are diagrams illustrating secondary structures ofan MS2SL motif and an Fr15 motif FIG. 12(A) illustrates a secondarystructure of an MS2 stem-loop motif, that is, an RNA motif to which theMS2 coat protein specifically binds (SEQ ID NO:4). FIG. 12(B)illustrates a secondary structure of an Fr15 motif, that is, an RNAmotif to which the Bacillus ribosomal protein S15 binds (SEQ ID NO:5).

FIG. 13 is a graph illustrating relationships, obtained in an mRNAhaving a 5′UTR regulation structure comprising a spacer and an MS2SLmotif, among the number of MS2SL motifs, the length of the spacer (i.e.,a distance of the MS2SL motif from the 5′ terminus) and thetranslational efficiency.

FIG. 14 is a graph illustrating relationships, obtained in an mRNAhaving a 5′UTR regulation structure comprising a spacer and an Fr15motif, among the number of Fr15 motifs, the length of the spacer (i.e.,a distance of the Fr15 motif from the 5′ terminus) and the translationalefficiency.

FIG. 15 is a conceptual diagram of a structure of a kink-turn RNA motifand a translation OFF switch system of the prior art using a target mRNAinto which the kink-turn RNA motif is incorporated (SEQ ID NO:84).

FIG. 16 is a conceptual diagram illustrating a conventional system forequally regulating transcription levels of multiple target genes in acell by using a combination of effector molecules and transcriptionfactor, which reveals that the activity of the transcription factor isdetermined depending upon the concentration of effector molecules.

FIGS. 17(A) and 17(B) are conceptual diagrams of an ON switch cassette,wherein FIG. 17(A) illustrates that a bait ORF is translated but a genefollowing an IRES sequence is not translated in the absence of a triggerprotein, and FIG. 17(B) illustrates that a bait ORF is not translatedbut a gene following the IRES sequence is translated owing to thefunction of a trigger protein in the presence of the trigger protein.

FIGS. 18(A), 18(B), 18(C) and 18(D) illustrate microphotographs of DsRed(expression of a trigger protein) and EGFP (translational object) inHeLa cells into which combinations each of a Kt motif or a dKt motif(negative control) and L7A or MS2 coat protein (negative control) as atrigger protein is introduced in the ON switch cassette, FIG. 18(E) is agraph illustrating a fluorescent ratio of EGFP to DsRed in eachcombination, and FIG. 18(F) is a graph illustrating mRNA level attainedby each combination.

FIG. 19(A) is a graph illustrating fluorescent intensity of EGFP againsta quantity ratio between mRNA plasmid and trigger protein plasmid ineach combination of an ON switch cassette or an OFF switch cassette anda Kt motif or an Fr15 motif, FIG. 19(B) is a graph illustratingfluorescent intensity of EGFP against a dissociation constant (Kd) ineach combination of the ON switch cassette or the OFF switch cassetteand each of Kt motif variants, and FIGS. 19(C) and (D) shows the resultof western blotting analysis for evaluating transcription activity ofcognate (L7Ae and S15, respectively) and noncognate (MS2CP and L7KK,respectively) input proteins.

FIG. 20(A) is a conceptual diagram of a switch-inverting module with orwithout a premature termination codon (PTC) (ON or ONn) (shown in left),and a graph illustrating a fluorescent intensity of EGFP from switchesinverted (shown in right), FIG. 20(B) shows the result of westernblotting analysis after siRNA-induced knocking down of nonsensemutation-dependent mRNA decay mechanism (NMD) factors: SMG1, UPF1 andUPF2, wherein GAPDH was also analyzed as an internal control of thelysates, and FIG. 20(C) is a set of graphs illustrating the meanintensity of EGFP fluorescence in siRNA-treated cells introduced witheach inverted switches (ON-Kt or ON-dKt) and trigger protein (MS2 orL7Ae).

FIG. 21(A) is a conceptual diagram of a series of switch-invertingmodules modified in bait ORF (320 nt (ON32), 160 nt (ON16) or 80 nt(ON8), which means length between PTC and the spliced site) (shown inleft) and a graph illustrating the mean intensity of EGFP fluorescencein the cells introduced with each inverted switches and trigger protein(shown in right), and FIG. 21(B) is a conceptual diagram ofswitch-inverting modules having a short chimeric intron (133 nt, ONc)and a graph illustrating the mean intensity of EGFP fluorescence in thecells introduced with the inverted switches (ONc-Kt or ONc-dKt) andtrigger protein (MS2 or L7Ae).

FIG. 22 is a set of graphs of flow cytometry analysis for the HeLa cellsintroduced with inverted switch (ON switch, ON-Kt (shown in left) or itsparental OFF switch, OFF-Kt (shown in right)) and trigger protein (L7Aeor its variants (L7-K or L7-KK) or negative control (N. C.)).

FIG. 23(A) is a set of graphs illustrating the mean intensity of EGFPfluorescence in the cells introduced with each amount of the plasmids(100 ng, 50 ng or 25 ng) expressing inverted switches (ON-Kt or ON-dKt),and FIG. 23(B) is a set of graphs illustrating the mean intensity ofEGFP fluorescence in the cells introduced inverted switches with theplasmids having different promoters (CMV (ON-Kt or ON-dKt), RSV promoter(R-ON-Kt or R-ON-dKt) or EF1α promoter (E-ON-Kt or E-ON-dKt)).

FIG. 24(A) is a conceptual diagram of plasmid expressing anti-apoptoticgene, Bcl-xL controlled by OFF switch and plasmid expressing apoptoticgene, Bim-EL instead of EGFP controlled by ON switch (ON-Kt-B), and FIG.24(B) is a graph of flow cytometry analysis for induction of Annexin Vpositive cells after inducing the each ON switch (ON-Kt-B or ON-dKt-B)and trigger protein (MS2 or L7Ae).

FIG. 25(A) is a graph illustrating the mean intensity of EGFP or ECFPexpressed by introducing ON or OFF switches together with or withoutL7Ae, FIG. 25(B) is a set of graphs illustrating the mean intensity ofEGFP or ECFP expressed by introducing ON (Fr15 and/or Kt) and OFF (dFr15and/or dKt) switches together with or without L7Ae and/or S15, FIG.25(C) is a conceptual diagram of the module (ON2) comprising of EGFPwith PTC as a bait-ORF instead of Renilla luciferase, and FIG. 25(D) isa graph of luciferases activity of cells introduced with ON or OFFswitch (ON2-Kt or ON2-dKt, respectively) and trigger protein (MS2 orL7Ae).

DESCRIPTION OF EMBODIMENTS

The present invention will now be described in detail with reference toembodiments. It is noted that the present invention is not limited tothe following embodiments.

According to a first embodiment, the present invention relates to atranslational control method. The translational control method iscarried out by using an mRNA having a regulation structure in the 5′UTR.The 5′UTR regulation structure contains a spacer positioned on the 3′side of a cap structure and on the 5′ side of an initiation codon, andone or more RNA-protein interaction motif-derived nucleotide sequencespositioned on the 3′ side of the spacer and on the 5′ side of theinitiation codon. When the mRNA having the regulation structure in the5′UTR is introduced into a cell in the presence of a proteinspecifically binding to the RNA-protein interaction motif-derivednucleotide sequence contained in the regulation structure, thetranslation of a protein encoded by the mRNA can be controlled dependingupon the structural characteristics of the 5′UTR regulation structure.Herein, an RNA-protein interaction motif-derived nucleotide sequence ora variant thereof is referred to as an “RNA motif” Besides, a proteinspecifically binding to the RNA motif and functioning as anindispensable element in the translational regulation is also referredto as a “trigger protein.”

First, the mRNA having the regulation structure in the 5′UTR will bedescribed. The mRNA having the regulation structure in the 5′UTR of thisembodiment is an artificially prepared non-natural mRNA that has a capstructure at the 5′ terminus, has an initiation codon and is designed toencode a desired target protein gene, and this mRNA is translated in aeukaryote cell so that the target protein encoded by the mRNA can beexpressed.

A structure on the 3′ side of the initiation codon can be designed bythose skilled in the art on the basis of the structure of a freelyselected natural or non-natural known mRNA. Typically, the nucleotidesequence can be determined so as to have an initiation codon, an openreading frame and a poly A sequence in the 3′UTR.

In the mRNA having the regulation structure in the 5′UTR of the presentembodiment, the 5′UTR contains, at the 5′ terminus, 7-methylguanosine5′-phosphate corresponding to the cap structure. This is because it isan indispensable structure in eukaryotic mRNA translation.

The 5′UTR regulation structure contains at least one or more RNA-proteininteraction motif-derived nucleotide sequences or a variant thereof. Inthe present invention, an RNA-protein complex interaction motif-derivednucleotide sequence encompasses: a nucleotide sequence known as an RNAsequence in the RNA-protein interaction motif of a known naturalRNA-protein complex; and a nucleotide sequence corresponding to an RNAsequence in an artificial RNA-protein complex interaction motif obtainedby the in vitro evolution method. The RNA-protein complexes areassemblies of proteins and RNAs which are confirmed in vivo in largenumbers, and are 3D objects having complicated structures.

A natural RNA-protein complex interaction motif-derived nucleotidesequence is usually composed of approximately 10 to 80 bases and isknown to specifically bind to a particular amino acid sequence of aparticular protein in a noncovalent manner, i.e., through a hydrogenbond. Such a natural RNA-protein complex interaction motif-derivednucleotide sequence can be selected from Tables 1 and 2 below and thedatabase:http://gibk26.bse.kyutech.ac.jp/jouhou/image/dna-protein/rna/rna.htmlavailable on the website.

TABLE 1 0RNA Protein Kd Reference 5S RNA (Xenopus laevis 5R1 0.64 ± 0.10nM Nat Struct Biol. 1998 Jul; 5(7): 543-6 oocyte) 5S RNA (Xenopus laevis5R2 0.35 ± 0.03 nM Nat Struct Biol. 1998 Jul; 5(7): 543-6 oocyte) dsRNAB2 1.4 ± 0.13 nM Nat Struct Mol Biol. 2005 Nov; 12(11): 952-7 RNAsplicing motif with Fox-1 0.49 nM at EMBO J. 2006 Jan 11; 25(1): 163-73.UGCAUGU element 150 mM salt TGE GLD-1 9.2 ± 2 nM J Mol Biol. 2005 Feb11; 346(1): 91-104. sodB mRNA Hfq 1.8 nM EMBO J. 2004 Jan 28; 23(2):396-405. RyhB (siRNA) Hfq 1500 nM Annu Rev Microbiol. 2004; 58: 303-28mRNA HuD 0.7 ± 0.02 nM Nat Struct Biol. 2001 Feb; 8(2): 141-5 S domainof 7S RNA human SRP19 RNA. 2005 Jul; 11(7): 1043-50. Epub 2005 May 31Large subunit of SRP human SRP19 2 nM Nat Struct Biol. 2001 Jun; 8(6):515-20 RNA 23S rRNA L1 Nat Struct Biol. 2003 Feb; 10(2): 104-8 23S rRNAL11 Nat Struct Biol. 2000 Oct; 7(10): 834-7 5S rRNA L18 Biochem J. 2002May 1; 363(Pt 3): 553-61 23S rRNA L20 13 ± 2 nM J Biol Chem. 2003 Sep19; 278(38): 36522-30. Own mRNA site1 L20 88 ± 23 nM J Biol Chem. 2003Sep 19; 278(38): 36522-30. Own mRNA site2 L20 63 ± 23 nM Mol Microbiol.2005 Jun; 56(6): 1441-56 23S rRNA L23 J Biomol NMR. 2003 Jun; 26(2):131-7 5S rRNA L25 EMBO J. 1999 Nov 15; 18(22): 6508-21 Own mRNA L30 NatStruct Biol. 1999 Dec; 6(12): 1081-3. mRNA LicT EMBO J. 2002 Apr 15;21(8): 1987-97 Own mRNA MS2 coat 39 ± 5 nM FEBS J. 2006 Apr; 273(7):1463-75 Stem-loop RNA motif Nova-2 Cell. 2000 Feb 4; 100(3): 323-32 SL2Nucleocapsid 110 ± 50 nM J Mol Biol. 2000 Aug 11; 301(2): 491-511Pre-rRNA Nucleolin EMBO J. 2000 Dec 15; 19(24): 6870-81 p19 0.17 ± 0.02nM Cell. 2003 Dec 26; 115(7): 799-811 Box C/D L7Ae 0.9 ± 0.2 nM RNA.2005 Aug; 11(8): 1192-200.

TABLE 2 RNA Protein Kd Reference siRNA with the PAZ (PiWi Argonaut andNat Struct Biol. 2003 Dec; 10(12): 1026-32. characteristic Zwille)two-base 3′ overhangs dsRNA Rnase III Cell. 2006 Jan 27; 124(2): 355-66HIV-1 RRE (IIB) RR1-38 3-8 nM Nat Struct Biol. 1998 Jul; 5(7): 543-6 OwnmRNA S15 5 nM EMBO J. 2003 Apr 15; 22(8): 1898-908 16S rRNA S15 6 nM NatStruct Biol. 2000 Apr; 7(4): 273-277. Own mRNA S15 43 nM EMBO J. 2003Apr 15; 22(8): 1898-908 16S rRNA S4 6.5 μM in 4° C., J Biol Chem. 1979Mar 25; 254(6): 1775-7 1.7 nM in 42° C. 16S rRNA S4 18 μM J Biol Chem.1979 Mar 25; 254(6): 1775-7 16S rRNA S8 26 ± 7 nM J Mol Biol. 2001 Aug10; 311(2): 311-24 mRNA S8 200 nM RNA. 2004 Jun; 10(6): 954-64 mRNA SacY1400 nM EMBO J. 1997 Aug 15; 16(16): 5019-29 SnRNA Sm Cold Spring HarbSymp Quant Biol. 2006; 71: 313-20. tmRNA SmpB 21 ± 7 nM J Biochem(Tokyo). 2005 Dec; 138(6): 729-39 TD3 of tmRNA SmpB 650 nM J Biochem(Tokyo). 2005 Dec; 138(6): 729-39 U1 snRNA snRNP U1A 0.032 ± 0.007 nMNat Struct Biol. 2000 Oct; 7(10): 834-7 (salt dependence) S domain of 7SRNA SRP54 500 nM RNA. 2005 Jul; 11(7): 1043-50. TAR Tat 200-800 nMNucleic Acids Res. 1996 Oct 15; 24(20): 3974-81 BIV TAR Tat 1.3 nM or 8nM or 60 nM Mol Cell. 2000 Nov; 6(5): 1067-76 (Mg dependence) tRNA^(Thr)ThrRS 500 nM Nat Struct Biol. 2002 May; 9(5): 343-7 thrS mRNA operatorThrRS 10 nM Trends Genet. 2003 Mar; 19(3): 155-61 Single stranded mRNATIS11d Nat Struct Mol Biol. 2004 Mar; 11(3): 257-64. PSTVd Virp1 500 nMNucleic Acids Res. 2003 Oct 1; 31(19): 5534-43 RNA hairpin; Smaug Vts1p30 nM Nat Struct Mol Biol. 2006 Feb; 13(2): 177-8. recognition element(SRE) λ BoxB λ N 90 nM Cell. 1998 Apr 17; 93(2): 289-99

An artificial RNA-protein complex interaction motif-derived nucleotidesequence is the nucleotide sequence of an RNA in an RNA-proteininteraction motif of an artificially designed RNA-protein complex. Sucha nucleotide sequence is usually composed of approximately 10 to 80bases and is designed to specifically bind to a particular amino acidsequence of a particular protein in a noncovalent manner, i.e., througha hydrogen bond. An example of such an artificial RNA-protein complexinteraction motif-derived nucleotide sequence includes an RNA aptamerspecifically binding to a particular protein. An RNA aptamerspecifically binding to a desired target protein can be obtained by, forexample, an evolutionary engineering method known as the in vitroselection method or the SELEX method. A trigger protein used in thiscase is a protein to which the RNA aptamer binds. For example,nucleotide sequences listed in Table 3 below are known, and thesenucleotide sequences may be also used as the RNA-protein complexinteraction motif-derived nucleotide sequence of the present invention.

TABLE 3 RNA Protein Kd Reference Rev aptamer 5 Rev 190 nM RNA. 2005 Dec;11(12): 1848-57 Aptamer p50 5.4 ± 2.2 nM Proc Natl Acad Sci USA. 2003Aug 5; 100(16): 9268-73. BMV Gag aptamer BMV Gag 20 nM RNA. 2005 Dec;11(12): 1848-57 BMV Gag aptamer CCMV Gag 260 nM RNA. 2005 Dec; 11(12):1848-57 CCMV Gag aptamer CCMV Gag 280 nM RNA. 2005 Dec; 11(12): 1848-57CCMV Gag aptamer BMV Gag 480 nM RNA. 2005 Dec; 11(12): 1848-57

In the present embodiment, as for the RNA-protein complex interactionmotif-derived nucleotide sequence, an RNA-protein complex correspondingto the origin of the nucleotide sequence preferably has a dissociationconstant Kd of approximately 0.1 nM to approximately 1 μM.

Furthermore, in addition to the RNA-protein complex interactionmotif-derived nucleotide sequence itself, a variant of such a sequenceis also encompassed by the RNA motif of the present invention. In thepresent invention, a variant refers to a variant having a dissociationconstant Kd not less than 10%, 20%, 30%, 40% or 50% or not more than10%, 20%, 30%, 40% or 50% from a protein specifically binding to theRNA-protein interaction motif-derived nucleotide sequence. Such avariant can be appropriately selected and used so as to attain a desiredtranslational level of the mRNA containing the RNA-protein complexinteraction motif. Here, attention should be paid to the fact thattranslational efficiency from an mRNA having a motif with a higherdissociation constant Kd is increased and the translational efficiencydecreases as the dissociation constant Kd decreases. Furthermore, thenucleotide sequence of such a variant may be one that can be hybridized,under stringent conditions, to a nucleic acid (corresponding to acomplementary strand) having a complementary sequence with theRNA-protein interaction motif-derived nucleotide sequence (correspondingto a positive strand). Here, the stringent conditions can be determinedon the basis of a melting temperature (Tm) of the nucleic acid to bebound as is taught by Berger and Kimmel (1987, Guide to MolecularCloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, SanDiego Calif.). For example, as conditions for washing afterhybridization, conditions of about “1×SSC, 0.1% SDS and 37° C.” can begenerally employed. The complementary strand is preferably retained tobe hybridized to the corresponding positive strand even when it iswashed under such conditions. Although not particularly limited, as morestringent hybridization conditions, the hybridized state between thepositive strand and the complementary strand can be retained underwashing conditions about “0.5×SSC, 0.1% SDS and 42° C.”, and even morestringent, under conditions about “0.1×SSC, 0.1% SDS and 65° C.”.Specifically, the variant has a nucleotide sequence with sequenceidentity of at least 90%, preferably, at least 95%, 96%, 97%, 98% or 99%with the RNA-protein interaction motif-derived nucleotide sequence. Sucha variant can maintain a constant binding with the protein specificallybinding to the RNA-protein interaction motif-derived nucleotidesequence, so as to make a contribution to translational repression.

Specific examples of the RNA motif of the present embodiment include5′-GGGCGUGAUGCGAAAGCUGACCC-3′ (SEQ ID NO: 1), that is, a nucleotidesequence to which L7Ae is known to be related to RNA modification suchas RNA methylation or pseudouridylation (Moore T et. al., Structure Vol.12, pp. 807-818 (2004)) binds, and its variants, kink-loop (SEQ ID NO:2) and kink-loop2 (SEQ ID NO: 3).

Other specific examples include MS2 stem-loop motif, that is, an RNAmotif to which an MS2 coat protein specifically binds (22: Keryer-BibensC, Barreau C, Osborne H B (2008) Tethering of proteins to RNAs bybacteriophage proteins. Biol Cell 100: 125-138) (SEQ ID NO: 4), andFr15, that is, an RNA motif to which Bacillus ribosomal protein S15binds (24: Batey R T, Williamson J R (1996) Interaction of the Bacillusstearothermophilus ribosomal protein S15 with 16 S rRNA: I. Defining theminimal RNA site. J Mol Biol 261: 536-549) (SEQ ID NO: 5).

Still other specific examples include5′-GGCGUAUGUGAUCUUUCGUGUGGGUCACCACUGCGCC-3′ (SEQ ID NO: 6), that is, anucleotide sequence to which threonyl-tRNA synthetase, an enzyme foraminoacylation, binding to its own mRNA and known to have a feedbackinhibition function to inhibit translation binds (Cell (Cambridge,Mass.) v97, pp. 371-381 (1999)), and a variant thereof. Still otherspecific examples includeR9-2;5′-GGGUGCUUCGAGCGUAGGAAGAAAGCCGGGGGCUGCAGAUAAUGUAUAGC-3′ (SEQ IDNO: 7), that is, an RNA-protein complex interaction motif-derivednucleotide sequence derived from Bcl-2 family CED-9, a cancercell-specific intrinsic protein, a variant thereof, a nucleotidesequence derived from an aptamer of an RNA sequence binding to NF-kappaB and a variant thereof.

In the present embodiment, the spacer contained in the 5′UTR regulationstructure if necessary is a portion composed of one or more bases and ispositioned between the cap structure at the 5′ terminus and the RNAmotif. The nucleotide sequence of the spacer is not particularly limitedbut may be a freely chosen sequence, and is preferably a nucleotidesequence that does not form a particular secondary or tertiarystructure, specifically, a nucleotide sequence that does not include aninitiation codon and does not encode a particular gene. In the presentembodiment, the spacer may be omitted, and hereinafter, a case in whichthe spacer is omitted is described as a case in which the number ofbases of the spacer is 0.

In the present embodiment, the 5′UTR of the mRNA contains the capstructure, the spacer and the RNA motif arranged in this order from the5′ terminus. FIG. 1 is a conceptual diagram of the 5′UTR regulationstructure with the cap structure omitted.

The spacer is placed immediately 3′ to the cap structure. Herein,“immediately” means that there is no base between the cap structure andthe spacer, but 1 to 6 technically necessary bases, such as arestriction enzyme site, may be placed therebetween in some cases.

The length of the spacer may be a freely chosen number of bases inaccordance with desired level of translation of mRNA, and the spacer mayhave, for example, 0 to 1000 bases, 0 to 900 bases, 0 to 800 bases, 0 to700 bases, 0 to 600 bases, 0 to 500 bases, 0 to 450 bases, 0 to 400bases, 0 to 350 bases, 0 to 300 bases, 0 to 250 bases, 0 to 200 bases, 0to 150 bases, 0 to 100 bases, 0 to 50 bases, 0 to 40 bases, 0 to 30bases, 0 to 20 bases or 0 to 10 bases, and preferably has 0 to 350bases. A nucleotide sequence indispensable in the embodiment, such as arestriction enzyme site, may be regarded as a part of the nucleotidesequence of the spacer. A spacer having a smaller number of bases showsa larger translational repression effect, so as to obtain an mRNA withlower translational efficiency. By increasing/decreasing the number ofbases of the spacer, an mRNA having translational efficiencysubstantially continuously regulated can be designed.

Examples of the spacer include sequences listed in Table 4.

TABLE 4 SEQ Base ID length Sequences NO. 18 UCAGAUCCGCUAGGAUCU  8 32UCAGAUCCGCUAGCGCUACCGGACUCAGAUCU  9 51UCAGAUCCGCUAGCCGCCUGUUUUGACCGCUGGGAU 10 CUGCCAUUGAGAUCU 67UCAGAUCCGCUAGCCCGACCGCCUUACUGCCGCCUG 11 UUUUGACCGCUGGGAUCUGCCAUUGAGAUCU94 UCAGAUCCGCUAGCUCGGAUUAGGGCCGCAAGAAAA 12CUAUCCCGACCGCCUUACUGCCGCCUGUUUUGACCG CUGGGAUCUGCCAUUGAGAUCU 120UCAGAUCCGCUAGCGCAGGUAGCAGAGCGGGUAAAC 13UGGCUCGGAUUAGGGCCGCAAGAAAACUAUCCCGACCGCCUUACUGCCGCCUGUUUUGACCGCUGGGAUCUG CCAUUGAGAUCU 145UCAGAUCCGCUAGCGGAUUGGCCUGAACUGCCAGCU 14GGCGCAGGUAGCAGAGCGGGUAAACUGGCUCGGAUUAGGGCCGCAAGAAAACUAUCCCGACCGCCUUACUGCCGCCUGUUUUGACCGCUGGGAUCUGCCAUUGAGAUC U 164UCAGAUCCGCUAGCGAUACACCGCAUCCGGCGCGGA 15UUGGCCUGAACUGCCAGCUGGCGCAGGUAGCAGAGCGGGUAAACUGGCUCGGAUUAGGGCCGCAAGAAAACUAUCCCGACCGCCUUACUGCCGCCUGUUUUGACCGCU GGGAUCUGCCAUUGAGAUCU 320UCAGAUCCGCUAGCGAUACACCGCAUCCGGCGCGGA 16UUGGCCUGAACUGCCAGCUGGCGCAGGUAGCAGAGCGGGUAAACUGGCUCGGAUUAGGGCCGCAAGAAAACUAUCCCGACCGCCUUACUGCCGCCUGUUUUGACCGCUGGGAUCUGCCAUUGAGAUCCGAUCCCGUCGUUUUACAACGUCGUGACUGGGAAAACCCUGGCGUUACCCAACUUAAUCGCCUUGCAGCACAUCCCCCUUUCGCCAGCUGGCGUAAUAGCGAAGAGGCCCGCACCGAUCGCCCUU CCCAACAGUUGCGCAGCCUGACCGGUAGAUCU

The one or more RNA motifs are placed immediately 3′ of the spacer.Also, “immediately” refers to a case in which 1 to 6 bases may be placedbetween the spacer and the RNA motif, but a nucleotide sequence providedbetween the spacer and the RNA motif is preferably regarded as a part ofthe spacer. If the 5′UTR regulation structure contains a plurality ofRNA motifs, one RNA motif may “immediately” follow an adjacent RNAmotif. Furthermore, it is preferable to design it so that a sequence ofapproximately 1 to 6 bases may be present as, for example, a restrictionenzyme site between the RNA motif positioned closest to the 3′ terminusand the initiation codon. The structure can be designed so as to inserta freely chosen number of RNA motifs on the 3′ side of the spacer. Forexample, the number of RNA motifs may be 1 to 8, or particularly 1 to 4,but the number of RNA motifs is not limited to such a particular number.

As the number of inserted RNA motifs increases, the translationalrepression effect for the target protein encoded by the mRNA increases,and hence, the translational efficiency decreases. Accordingly, the mRNAtranslational efficiency is decreased, on the order of an mRNA having a5′UTR regulation structure comprising one RNA motif, an mRNA having a5′UTR regulation structure comprising two RNA motifs, an mRNA having a5′UTR regulation structure comprising three RNA motifs and an mRNAhaving a 5′UTR regulation structure comprising four RNA motifs, as longas the rest of the 5′UTR structure is the same.

In the 5′UTR regulation structure, the translational efficiency can beregulated mainly depending upon the number of RNA motifs. Thistranslational efficiency is discretely regulated by increasing ordecreasing the number of RNA motifs. The number of RNA motifs is, forexample, 1 to 20, preferably 1 or more or 2 or more, and 20 or less, 10or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, or 4 orless.

As described above, the total base length of the 5′UTR of the mRNA ofthe present invention is determined depending upon the length of thespacer and the number of RNA motifs. However, it is not preferable thatthe 5′UTR of the mRNA of the present embodiment form a complicatedtertiary structure in the whole region. This is because thetranslational regulation is made to function when a trigger proteinbinds to the RNA motif, and hence, if a tertiary structure to which atrigger protein cannot bind is formed, there is a possibility that theregulation function cannot be secured. The possibility of the 5′UTR of adesigned mRNA forming a complicated tertiary structure can be calculatedby using, on a computer, appropriate software, such as Discovery Studioor Centrolid Fold. Then, the mRNA can be designed so as to prevent the5′UTR from forming such a complicated tertiary structure. Alternatively,after actually preparing an mRNA, binding between the mRNA and a triggerprotein may be confirmed in vitro, or translation obtained in theabsence of a trigger protein may be confirmed in a cell, so as toconfirm whether or not the designed structure is a structure in whichthe translational regulation cannot work because the trigger proteincannot bind thereto.

In the mRNA having the regulation structure in the 5′UTR of the presentembodiment, the translation is decreased and the translationalefficiency is lower in the presence of a trigger protein as comparedwith an mRNA encoding the same gene but having no regulation structurein the 5′UTR. On the other hand, in the of absence of the triggerprotein, the translational efficiency is substantially equivalent in themRNA having the regulation structure in the 5′UTR and the mRNA encodingthe same gene but having no regulation structure in the 5′UTR. WhenmRNAs have different sequences in the 5′UTR regulation structure, thetranslational efficiency is generally different. These characteristicsof mRNAs may be used for designing, for example, an mRNA capable ofachieving translational efficiency with a freely chosen value rangingbetween approximately 0% and approximately 99% in the presence of atrigger protein as compared with an mRNA having no regulation structurein the 5′UTR. The relationship among the translational efficiency, thenumber of bases of the spacer and the number of RNA motifs can bedetermined, for example, on the basis of a standard curve obtained bydesigning and preparing a plurality of mRNAs having different 5′UTRregulation structures and measuring translational efficiency of thesemRNAs. The translational regulation can be accurately performed bydesigning the structure of the 5′UTR by appropriately using the numberof bases of the spacer for achieving the aforementioned continuousregulation and the number of RNA motifs for achieving the discreteregulation.

In another embodiment, since the translational efficiency is determineddepending upon the number of bases of the spacer and the type and thenumber of RNA motifs, mRNAs having 5′UTRs containing a large number ofregulation structures are prepared by combining these variable elements.A combination achieving desired translational efficiency is selectedfrom the employed combinations, so that an mRNA having a regulationstructure in the 5′UTR and realizing desired translational efficiencycan be obtained.

When a freely chosen mRNA having a regulation structure in the 5′UTR canbe designed and its nucleotide sequence can be determined, those skilledin the art can prepare such an mRNA by any of a plurality ofconventional gene engineering methods. An example of the methodsincludes a method in which an mRNA is expressed in a cell by using anexpression vector. This method can be performed by introducing, into acell, an expression vector having a sequence in which a promoter capableof functioning in a host cell and an mRNA having a regulation structurein the 5′UTR are functionally concatenated. As the expression vector,for example, virus vectors such as a retrovirus, lentivirus, adenovirus,adeno-associated virus, herpes virus and Sendai virus vectors, or animalcell expression plasmids (such as pA1-11, pXT1, pRc/CMV, pRc/RSV andpcDNAI/Neo) can be used. Furthermore, as the promoter, an EF-α promoter,a CAG promoter, an SRα promoter, an SV40 promoter, an LTR promoter, aCMV (cytomegalovirus) promoter, an RSV (Rous sarcoma virus) promoter, anMoMuLV (Moloney murine leukemia virus) LTR, an HSV-TK (Herpes simplexvirus thymidine kinase) promoter or the like can be used. Especially, anEF-α promoter, a CAG promoter, an LTR and a CMV promoter can be suitablyused. Furthermore, the expression vector may appropriately contain anenhancer, a selectable marker gene, SV40 replication origin and thelike. Examples of the selectable marker gene include a dihydrofolatereductase gene, a hygromycin resistance gene, a Blasticidin resistancegene, a neomycin resistance gene and a puromycin resistance gene. Asother exemplary methods, if the mRNA is directly introduced into a cellwithout using an expression vector, the mRNA can be introduced into acell by a lipofection method, a liposomal method, an electroporationmethod, a calcium phosphate transfection method, a DEAE dextran method,a microinjection method, a gene gun method or the like. An mRNA can besynthesized from a template DNA having a desired mRNA sequence by theRNA polymerase method, and the synthesized RNA is provided, at the 5′terminus, with a cap structure by using, for example, m7G cap analogue(Promega), so as to be used as the mRNA of the present embodiment.

A translational regulation method for a target protein in a cell byusing the thus prepared mRNA may be performed by following method. Thedescribed mRNA having the regulation structure in the 5′UTR plays afunctional role in the presence of the trigger protein specificallybinding to the RNA motif contained in the regulation structure. In thismethod, it is necessary to introduce, into a cell, the mRNA having theregulation structure in the 5′UTR and the protein specifically bindingto the RNA motif contained in this regulation structure. Accordingly, inone embodiment, the method for regulating translation of a protein in acell comprises a step of introducing, into a cell, an mRNA having aregulation structure in the 5′UTR and a protein specifically binding toan RNA motif contained in the regulation structure.

The step of introducing the mRNA having the regulation structure in the5′UTR into a cell can be carried out by introducing an expression vectorfor expressing the mRNA or the mRNA itself by any of the aforementionedmethods. The amount introduced is varied depending on the cell forintroduction, the introducing method and the type of introductionreagent, and those skilled in the art can appropriately select them forattaining a desired translational level.

The trigger protein is determined in accordance with the RNA motif. Sucha trigger protein is not particularly limited, and examples of theprotein include those listed in Tables 1 to 3. The trigger protein ispreferably a protein intrinsically expressed in a cell of interest. Thetrigger protein may be a fusion protein fused with another proteinhaving a different function as long as it contains a proteinspecifically binding to the RNA motif contained in the 5′UTR regulationstructure. The step of introducing the trigger protein into a cell canbe carried out by introducing, into a cell, an expression vector havinga gene encoding the protein or the protein fused with a proteintransduction domain (PTD) or a cell penetrating peptide (CPP). Examplesof the PTD include drosophila-derived AntP, HIV-derived TAT (Frankel, A.et al., Cell 55, 1189-93 (1988) or Green, M. & Loewenstein, P. M. Cell55, 1179-88 (1988)), Penetratin (Derossi, D. et al., J. Biol. Chem. 269,10444-50 (1994)), Buforin II (Park, C. B. et al., Proc. Natl Acad. Sci.USA 97, 8245-50 (2000)), Transportan (Pooga, M. et al., FASEB J. 12,67-77 (1998)), MAP (model amphipathic peptide) (Oehlke, J. et al.,Biochim. Biophys. Acta. 1414, 127-39 (1998)), K-FGF (Lin, Y. Z. et al.,J. Biol. Chem. 270, 14255-14258 (1995)), Ku70 (Sawada, M. et al., NatureCell Biol. 5, 352-7 (2003)), Prion (Lundberg, P. et al., Biochem.Biophys. Res. Commun. 299, 85-90 (2002)), pVEC (Elmquist, A. et al.,Exp. Cell Res. 269, 237-44 (2001)), Pep-1 (Morris, M. C. et al., NatureBiotechnol. 19, 1173-6 (2001)), Pep-7 (Gao, C. et al., Bioorg. Med.Chem. 10, 4057-65 (2002)), SynB1 (Rousselle, C. et al., Mol. Pharmacol.57, 679-86 (2000)), HN-I (Hong, F. D. & Clayman, G L. Cancer Res. 60,6551-6 (2000)), and one using a cell penetrating domain of a proteinsuch as HSV-derived VP22. An example of the CPP includes polyargininesuch as 11R (Cell Stem Cell, 4, 381-384 (2009)) or 9R (Cell Stem Cell,4, 472-476 (2009)). In order to increase/decrease the amount of triggerprotein expressed in the cell, the number of plasmid vectors to beintroduced can be altered, so that the translation of the mRNA havingthe regulation structure in the 5′UTR can be further controlled.Although not particularly limited, the plasmid vector expressing thetrigger protein can be introduced into the cell so that a ratio betweenthe mRNA having the regulation structure in the 5′UTR and the triggerprotein can be, for example, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

In the present invention, if an ON switch cassette is placed on the 5′side of an initiation codon of an mRNA gene and on the 3′ side of an RNAmotif, the translational repression can be reversed so as to control themRNA translation to be carried out merely in the presence of a triggerprotein. An ON switch cassette is composed of a sequence comprising,from the 5′ side, a variant open reading frame (a bait ORF), intron andan internal ribosome entry site (IRES) in this order. A bait ORF is avariant ORF having, in a sequence encoding a freely chosen gene, a stopcodon in more than 320 bases from the 3′ end binding to intron forcausing RNA decay by nonsense mutation-dependent mRNA decay mechanism(NMD). In the present invention the bait ORF may be any cording gene. Anexample of the bait ORF includes, but is not especially limited to, asequence in which a stop codon is inserted at the 457th and/or 466thbase from the 5′ side of Renilla luciferase (SEQ ID NO: 17 or 81) or asequence in which a stop codon is inserted at the 172th base from the 5′side of EGFP (SEQ ID NO: 82). Furthermore, the intron may have asequence to which spliceosome binds, and an example includes a sequenceof 20 or more bases containing a GT sequence on the 5′ end and an AGsequence on the 3′ end. Preferably, the intron is human β globin intron(SEQ ID NO: 18) or chimeric intron (SEQ ID NO: 83).

Such an mRNA having the 5′UTR regulation structure comprising the ONswitch cassette can be introduced into a cell, so that the translationof the target protein can be started by the trigger protein.Furthermore, the level of translation can be regulated by appropriatelycontrolling the spacer and the number of RNA motifs in the 5′UTRregulation structure as described above.

In one embodiment of the present invention, the translational regulationfor multiple mRNAs different in the 5′UTR regulation structure and inthe target protein to encode can be simultaneously carried out in thepresence of a single trigger protein. In this translational regulationmethod, multiple mRNAs each having a different 5′UTR regulationstructure and having a different target protein gene are first designed.A case of simultaneous translational regulation for two mRNAs will beexemplarily described, but simultaneous translational regulation forthree, four, five or more mRNAs can be performed. Such simultaneoustranslational regulation can be carried out by designing and preparingmultiple mRNAs by a design method similar to that described below andintroducing the resulting mRNAs into a cell.

The first mRNA to be designed may have a nucleotide sequence having acap structure at the 5′ terminus and encoding a first target proteingene. A first 5′UTR regulation structure of the first mRNA is designedto contain a spacer of 0 to 350 bases and one or more first RNA motifshaving an RNA-protein interaction motif-derived nucleotide sequence or avariant thereof. The second mRNA similarly having a nucleotide sequencehaving a cap structure at the 5′ terminus and encoding a second targetprotein gene, and a second 5′UTR regulation structure of the second mRNAis also designed to contain a spacer of 0 to 350 bases and one or moresecond RNA motifs having an RNA-protein interaction motif-derivednucleotide sequence or a variant thereof. Thereafter, these two mRNAsare simultaneously introduced into a cell, so that the first targetprotein gene and the second target protein gene can be obtained asdifferent genes desired to be expressed.

Preparation of an induced pluripotent stem cell (an iPS cell) byintroducing four protein genes into a somatic cell will be exemplarilydescribed according to Papapetrou E P et al. (2009) Stoichiometric andtemporal requirements of Oct4, Sox2, Klf4, and c-Myc expression forefficient human iPSC induction and differentiation. Proc Natl Acad SciUSA 106: 12759-12764. A gene encoding Oct3/4 protein is used as a firsttarget protein gene, a gene encoding Sox2 protein is used as a secondtarget protein gene, a gene encoding Klf4 protein is used as a firsttarget protein gene and a gene encoding c-Myc protein is used as a firsttarget protein gene. These genes are designed so that the level oftranslation of the Oct3/4 protein can be about three times as high asthat of the other three proteins by the method described above, and thethus designed genes are introduced into a somatic cell. In this manner,the preparation efficiency for the iPS cell can be advantageouslyincreased.

The first RNA motif and the second RNA motif may be the same.Alternatively, the first RNA motif and the second RNA motif may be in avariant relationship in which they specifically bind to the same triggerprotein but have different dissociation constants. This is because thetranslational regulation should function in the presence of a singletrigger protein.

The numbers of the first RNA motifs and the second RNA motifs may be thesame or different, and the numbers and the types of sequences of thespacers may be the same or different. The first 5′UTR regulationstructure and the first 5′UTR regulation structure, however, should havedifferent structures as a whole for achieving different translationalefficiencies. The 5′UTR regulation structures for attaining desiredtranslational efficiency can be appropriately designed by those skilledin the art by following the disclosure of the present application.

When the first mRNA and the second mRNA are designed, they are preparedby an ordinary method, and the prepared mRNAs are introduced into a cellexpressing a trigger protein or introduced simultaneously with a triggerprotein or genes expressing thereof into the same cell. In this manner,the translational regulation method can be carried out.

In the conventional technique, the expression level of an exogenous geneis regulated by controlling the type and the introduction amount of apromoter. When the present embodiment is employed, however, an exogenousgene can be stably expressed in a cell at a desired level oftranslation. Therefore, in the field of, for example, reprogrammingtechniques for expressing exogenous genes in cells in which optimumexpression level of a gene is demanded, optimum cell transformation canbe advantageously achieved through the reprogramming by regulatingdesired gene expression level in multiple stages.

In another embodiment, the present invention provides a method forselecting an exogenous mRNA translating a protein in a cell at a freelyselected level. This method comprises the steps of (1) introducing anyone of the aforementioned mRNAs having the 5′UTR regulation structuresinto a cell expressing a protein specifically binding to a correspondingRNA motif; and (2) measuring the level of translation of the protein toidentify the mRNA achieving a desired translational level.

The step (1) can be performed, as described in the first embodiment, bydesigning any one of the mRNAs having the 5′UTR regulation structuresand introducing the mRNA into a cell with an expression vector used ordirectly into a cell without using an expression vector. All of thedescription exemplarily given in the first embodiment is applicable alsoin this embodiment.

The step (2) can be performed by measuring the amount of the proteintranslated by the mRNA. The measurement of the amount of protein can becarried out by labeling the protein with an antibody to the desiredprotein or, if the protein to be translated is a fluorescent protein, byusing the fluorescence, by a method known to those skilled in the art,such as ELISA (enzyme-linked immunosorbent assay), EIA (enzymeimmunoassay), RIA (radioimmunoassay), Western blotting or flowcytometry.

The present invention will now be described in more details withreference to examples. It is noted that the present invention is notlimited to the following examples.

EXAMPLE 1

[Measurement of Translational Efficiency of Sp-Kt-EGFP, Kt-Sp-EGFP andKt-EGFP]

Three mRNAs encoding the gene of EGFP were designed by providing, in the5′UTR, a kink-turn RNA motif, that is, a binding RNA motif of anarchaeal ribosomal protein, L7Ae protein. The kink-turn RNA motif ishereinafter referred to as the Kt motif FIGS. 2(A) and 2(B) illustratethe outline of the design. FIG. 2(A) shows a structure, in the 5′UTR ofthe mRNA, containing a cap structure, a spacer and the Kt motif arrangedfrom the 5′ terminus, which structure is herein referred to asSp-Kt-EGFP. FIG. 2(B) shows a structure, in the 5′UTR of the mRNA,containing a cap structure, the Kt motif and a spacer arranged from the5′ terminus, which structure is herein referred to as Kt-Sp-EGFP.Although not shown in the drawing, an mRNA containing, in the 5′UTR, acap structure and the Kt motif arranged without providing a spacer wasalso designed. This structure is referred to as Kt-EGFP. The detailedsequences of the 5′UTRs of the Sp-Kt-EGFP, the Kt-Sp-EGFP and theKt-EGFP are shown in Table 5 below. By a method described in an item (1)below, plasmids expressing these mRNAs were prepared.

In accordance with a method described in an item (2) below, a plasmidexpressing L7Ae as a trigger protein was prepared, and the plasmidsexpressing the respective mRNAs were transfected into a cell and thetranslational efficiency was measured as described in items (3) to (5)below. As a result, although the expression was decreased in using theSp-Kt-EGFP and the Kt-EGFP, the Kt-Sp-EGFP did not give such an effect.It is understood from these results that the translational repressioneffect of the L7Ae is reduced from 0.019 to 0.23 because of the spacerpositioned between the 5′ terminus and the Kt motif.

EXAMPLE 2

[Preparation of mRNAs with Position of Kt Motif from 5′ TerminusAltered]

Next, mRNAs were designed by providing spacers so as to have the 5′terminal bases of Kt motifs positioned in the 18th, 51th, 67th, 94th,120th, 145th and 320th bases from the 5′ terminus. The number of Ktmotifs for each mRNA was one. The nucleotide sequences of the 5′UTRs ofthe respective mRNAs are shown in Table 5 below as constructs 18nt-Kt,51nt-Kt, 67nt-Kt, 94nt-Kt, 120nt-Kt, 145nt-Kt and 320nt-Kt. Plasmidsexpressing these mRNAs were prepared, so as to examine the translationalefficiencies. FIG. 3 illustrates the outline of the mRNA design. As aresult, in the presence of the L7Ae protein working as the triggerprotein specifically binding to the Kt motif, the translationalefficiency was found to be higher as the number of bases of the spacerwas greater, namely, as the distance of the Kt motif from the 5′terminus was greater. Conversely, it was found that as the Kt motif iscloser to the 5′ terminus, the translational repression is stronger.FIG. 4 illustrates a graph of the translational efficiency against thedistance of the Kt motif from the 5′ terminus.

As controls, mRNAs were prepared by replacing underlined Gs and As inthe nucleotide sequences of the 5′UTRs of the respective mRNAs of Table5 by C, as constructs 18nt-dKt, 51nt-dKt, 67nt-dKt, 94nt-dKt, 120nt-dKt,145nt-dKt and 320nt-dKt. It is known that the L7Ae protein cannot bindto mRNAs in which the underlined Gs and As are replaced by C. A dKtmotif is a construct obtained by inactivating the Kt motif. Thetranslational efficiency of these constructs was also examined in thepresence of the L7Ae protein by a similar method. The results areillustrated in FIG. 4 as a similar graph of the translationalefficiency. In each mRNA having the dKt motif inserted by replacing thetwo bases of the Kt motif by C, the translational repression was notcaused regardless of the distance of the dKt motif from the 5′ terminus.

TABLE 5 Trans- SEQ lational ID construct Sequences of the 5′ UTRefficiency NO Kt-EGFP UCAGAUCCGCUAGCGCUACCGGACUCAGA 0.019 ± 19 (32nt-Kt)UCUGGGGCGUGAUCCGAAAGGUGACCCG 0.0019 GAUCCACCGGUCGCCACCAUG Sp-Kt-EGFPUCAGAUCCGCUAGCGAUACACCGCAUCC 0.23 ± 20 (164nt-Kt)GGCGCGGAUUGGCCUGAACUGCCAGCUG 0.031 GCGCAGGUAGCAGAGCGGGUAAACUGGCUCGGAUUAGGGCCGCAAGAAAACUAUCC CGACCGCCUUACUGCCGCCUGUUUUGACCGCUGGGAUCUGCCAUUGAGAUCUGGGG CGUGAUCCGAAAGGUGACCCGGAUCCAC CGGUCGCCACCAUGKt-Sp-EGFP UCAGAUCCGCUAGCGCUACCGGACUCAGA 0.029 ± 21UCUGGGGCGUGAUCCGAAAGGUGACCCG 0.0042 GAUCCGAUCCCGUCGUUUUACAACGUCGUGACUGGGAAAACCCUGGCGUUACCCAA CUUAAUCGCCUUGCAGCACAUCCCCCUUUCGCCAGCUGGCGUAAUAGCGAAGAGGC CCGCACCGAUCGCCCUUCCCAACAGUUGCGCAGCCUGACCGGUCGCCACCAUG 18nt-Kt UCAGAUCCGCUAGGAUCUGGGGCGUGAU 0.017 ± 22CCGAAAGGUGACCCGGAUCCACCGGUCG 0.0047 CCACCAUG 51nt-KtUCAGAUCCGCUAGCCGCCUGUUUUGACC 0.026 ± 23 GCUGGGAUCUGCCAUUGAGAUCUGGGGC0.0065 GUGAUCCGAAAGGUGACCCGGAUCCACC GGUCGCCACCAUG 67nt-KtUCAGAUCCGCUAGCCCGACCGCCUUACU 0.036 ± 24 GCCGCCUGUUUUGACCGCUGGGAUCUGC0.0024 CAUUGAGAUCUGGGGCGUGAUCCGAAAG GUGACCCGGAUCCACCGGUCGCCACCAU G94nt-Kt UCAGAUCCGCUAGCUCGGAUUAGGGCCG 0.094 ± 25CAAGAAAACUAUCCCGACCGCCUUACUG 0.012 CCGCCUGUUUUGACCGCUGGGAUCUGCCAUUGAGAUCUGGGGCGUGAUCCGAAAGG UGACCCGGAUCCACCGGUCGCCACCAUG 120nt-KtUCAGAUCCGCUAGCGCAGGUAGCAGAGC 0.075 ± 26 GGGUAAACUGGCUCGGAUUAGGGCCGCA0.019 AGAAAACUAUCCCGACCGCCUUACUGCC GCCUGUUUUGACCGCUGGGAUCUGCCAUUGAGAUCUGGGGCGUGAUCCGAAAGGUG ACCCGGAUCCACCGGUCGCCACCAUG 145nt-KtUCAGAUCCGCUAGCGGAUUGGCCUGAAC 0.18 ± 27 UGCCAGCUGGCGCAGGUAGCAGAGCGGG0.032 UAAACUGGCUCGGAUUAGGGCCGCAAGA AAACUAUCCCGACCGCCUUACUGCCGCCUGUUUUGACCGCUGGGAUCUGCCAUUGA GAUCUGGGGCGUGAUCCGAAAGGUGACCCGGAUCCACCGGUCGCCACCAUG 320nt-Kt UCAGAUCCGCUAGCGAUACACCGCAUCC 0.24 ± 28GGCGCGGAUUGGCCUGAACUGCCAGCUG 0.040 GCGCAGGUAGCAGAGCGGGUAAACUGGCUCGGAUUAGGGCCGCAAGAAAACUAUCC CGACCGCCUUACUGCCGCCUGUUUUGACCGCUGGGAUCUGCCAUUGAGAUCCGAUC CCGUCGUUUUACAACGUCGUGACUGGGAAAACCCUGGCGUUACCCAACUUAAUCGC CUUGCAGCACAUCCCCCUUUCGCCAGCUGGCGUAAUAGCGAAGAGGCCCGCACCGA UCGCCCUUCCCAACAGUUGCGCAGCCUGACCGGUAGAUCUGGGGCGUGAUCCGAAA GGUGACCCGGAUCCACCGGUCGCCACCAU G

In Table 5, an initiation codon is shown in bold. Besides, BglII siteand BamHI site corresponding to both ends of the Kt motif are shown initalics. Each underlined base is a base replaced by C for inactivating aKt motif into a dKt motif.

It is revealed from these results that the binding between the mRNAencoding the target protein and the L7Ae working as the trigger proteinis indispensable to the translational regulation for the target proteinaccording to the present invention, and that a distance between the openreading frame and the 5′ terminus of the mRNA, namely, the spacer, isnot an indispensable element. It was also understood that thetranslational efficiency is increased substantially in proportion to thelength of the spacer even when the expression level (the abundance) ofthe L7Ae is constant. In other words, it was understood that thetranslation of the target protein can be quantitatively regulated inaccordance with the distance between the 5′ terminus of the mRNA and theRNA motif (the Kt motif).

EXAMPLE 3

[Two-Dimensional Approach]

It was found, as a result of Examples 1 and 2, that the translationalrepression attained by the Kt motif and the L7Ae is approximately 2% toapproximately 20%. If the Kt motif is away from the 5′ terminus of themRNA by 164 or more bases, further repression cannot be attained but thetranslational efficiency is approximately 20%. In this example, in orderto expand the translational regulatable range, an mRNA into which aK-loop RNA motif was inserted instead of the Kt motif was used. FIG.5(A) illustrates the structure of the K-loop RNA motif. The K-loop RNAmotif is hereinafter referred to as the Kl motif. It is known that theKl motif has binding affinity to the L7Ae approximately 500 times asweak as that of the Kt motif. Accordingly, the translational repressionin the presence of L7Ae attained by the mRNA containing the Kl motif isweaker than the translational repression in the presence of L7Aeattained by the mRNA under the same conditions except that the Kt motifis used instead of the Kl motif.

The present inventors prepared multiple mRNAs each containing the Klmotif FIGS. 6(A), 6(B) and 6(C) illustrate the outline of thepreparation. Specifically, an mRNA provided with one Kl motif (FIG.6(A)), an mRNA provided with two Kl motifs (FIG. 6(B)) and an mRNAprovided with three, four or more Kl motifs (FIG. 6(C)) were prepared.Furthermore, sixteen mRNAs were prepared by setting the distances of theKl motif positioned closest to the 5′ terminus from the 5′ terminusrespectively to 18 bases, 67 bases, 120 bases and 164 bases with theinitiation codon, the open reading frame and the structure of the 3′UTRset to be the same in these mRNAs. Table 6 below shows the details ofthese mRNAs. When multiple Kl motifs were contained, a distance betweenthe adjacent Kl motifs was set to 6 bases.

TABLE 6 Trans- lational SEQ ID construct Sequencesof the 5′ UTRefficiency NO 18nt-1xK1 UCAGAUCCGCUAGGAUCCGGGUGUGAAC 0.28 ± 29GGUGAUCACCCGAGAUCCACCGGUCGCC 0.071 ACCAUG 18nt-2xK1UCAGAUCCGCUAGGAUCCGGGUGUGAAC 0.084 ± 30 GGUGAUCACCCGAGAUCCGGGUGUGAAC0.014 GGUGAUCACCCGAGAUCCACCGGUCGCC ACCAUG 18nt-3xK1UCAGAUCCGCUAGGAUCCGGGUGUGAAC 0.039 ± 31 GGUGAUCACCCGAGAUCCGGGUGUGAAC0.013 GGUGAUCACCCGAGAUCCGGGUGUGAAC GGUGAUCACCCGAGAUCCACCGGUCGCC ACCAUG18nt-4xK1 UCAGAUCCGCUAGGAUCCGGGUGUGAAC 0.069 ± 32GGUGAUCACCCGAGAUCCGGGUGUGAAC 0.011 GGUGAUCACCCGAGAUCCGGGUGUGAACGGUGAUCACCCGAGAUCCGGGUGUGAAC GGUGAUCACCCGAGAUCCACCGGUCGCC ACCAUG67nt-1xK1 UCAGAUCCGCUAGCCCGACCGCCUUACU 0.41 ± 33GCCGCCUGUUUUGACCGCUGGGAUCUGC 0.060 CAUUGAGAUCCGGGUGUGAACGGUGAUCACCCGAGAUCCACCGGUCGCCACCAUG 67nt-2xK1 UCAGAUCCGCUAGCCCGACCGCCUUACU0.25 ± 34 GCCGCCUGUUUUGACCGCUGGGAUCUGC 0.027CAUUGAGAUCCGGGUGUGAACGGUGAUC ACCCGAGAUCCGGGUGUGAACGGUGAUCACCCGAGAUCCACCGGUCGCCACCAUG 67nt-3xK1 UCAGAUCCGCUAGCCCGACCGCCUUACU0.15 ± 35 GCCGCCUGUUUUGACCGCUGGGAUCUGC 0.015CAUUGAGAUCCGGGUGUGAACGGUGAUC ACCCGAGAUCCGGGUGUGAACGGUGAUCACCCGAGAUCCGGGUGUGAACGGUGAUC ACCCGAGAUCCACCGGUCGCCACCAUG 67nt-4xK1UCAGAUCCGCUAGCCCGACCGCCUUACU 0.12 ± 36 GCCGCCUGUUUUGACCGCUGGGAUCUGC0.018 CAUUGAGAUCCGGGUGUGAACGGUGAUC ACCCGAGAUCCGGGUGUGAACGGUGAUCACCCGAGAUCCGGGUGUGAACGGUGAUC ACCCGAGAUCCGGGUGUGAACGGUGAUCACCCGAGAUCCACCGGUCGCCACCAUG 120nt-1xK1 UCAGAUCCGCUAGCGCAGGUAGCAGAGC0.67 ± 37 GGGUAAACUGGCUCGGAUUAGGGCCGCA 0.059AGAAAACUAUCCCGACCGCCUUACUGCC GCCUGUUUUGACCGCUGGGAUCUGCCAUUGAGAUCCGGGUGUGAACGGUGAUCACC CGAGAUCCACCGGUCGCCACCAUG 120nt-2xK1UCAGAUCCGCUAGCGCAGGUAGCAGAGC 0.31 ± 38 GGGUAAACUGGCUCGGAUUAGGGCCGCA0.031 AGAAAACUAUCCCGACCGCCUUACUGCC GCCUGUUUUGACCGCUGGGAUCUGCCAUUGAGAUCCGGGUGUGAACGGUGAUCACC CGAGAUCCGGGUGUGAACGGUGAUCACCCGAGAUCCACCGGUCGCCACCAUG 120nt-3xK1 UCAGAUCCGCUAGCGCAGGUAGCAGAGC 0.19 ±39 GGGUAAACUGGCUCGGAUUAGGGCCGCA 0.015 AGAAAACUAUCCCGACCGCCUUACUGCCGCCUGUUUUGACCGCUGGGAUCUGCCAU UGAGAUCCGGGUGUGAACGGUGAUCACCCGAGAUCCGGGUGUGAACGGUGAUCACC CGAGAUCCGGGUGUGAACGGUGAUCACCCGAGAUCCACCGGUCGCCACCAUG 120nt-4xK1 UCAGAUCCGCUAGCGCAGGUAGCAGAGC 0.12 ±40 GGGUAAACUGGCUCGGAUUAGGGCCGCA 0.0052 AGAAAACUAUCCCGACCGCCUUACUGCCGCCUGUUUUGACCGCUGGGAUCUGCCAU UGAGAUCCGGGUGUGAACGGUGAUCACCCGAGAUCCGGGUGUGAACGGUGAUCACC CGAGAUCCGGGUGUGAACGGUGAUCACCCGAGAUCCGGGUGUGAACGGUGAUCACC CGAGAUCCACCGGUCGCCACCAUG 164nt-1xK1UCAGAUCCGCUAGCGAUACACCGCAUCC 0.58 ± 41 GGCGCGGAUUGGCCUGAACUGCCAGCUG0.087 GCGCAGGUAGCAGAGCGGGUAAACUGGC UCGGAUUAGGGCCGCAAGAAAACUAUCCCGACCGCCUUACUGCCGCCUGUUUUGAC CGCUGGGAUCUGCCAUUGAGAUCCGGGUGUGAACGGUGAUCACCCGAGAUCCACCG GUCGCCACCAUG 164nt-2xK1UCAGAUCCGCUAGCGAUACACCGCAUCC 0.34 ± 42 GGCGCGGAUUGGCCUGAACUGCCAGCUG0.044 GCGCAGGUAGCAGAGCGGGUAAACUGGC UCGGAUUAGGGCCGCAAGAAAACUAUCCCGACCGCCUUACUGCCGCCUGUUUUGAC CGCUGGGAUCUGCCAUUGAGAUCCGGGUGUGAACGGUGAUCACCCGAGAUCCGGGU GUGAACGGUGAUCACCCGAGAUCCACCG GUCGCCACCAUG164nt-3xK1 UCAGAUCCGCUAGCGAUACACCGCAUCC 0.21 ± 43GGCGCGGAUUGGCCUGAACUGCCAGCUG 0.021 GCGCAGGUAGCAGAGCGGGUAAACUGGCUCGGAUUAGGGCCGCAAGAAAACUAUCC CGACCGCCUUACUGCCGCCUGUUUUGACCGCUGGGAUCUGCCAUUGAGAUCCGGGU GUGAACGGUGAUCACCCGAGAUCCGGGUGUGAACGGUGAUCACCCGAGAUCCGGGU GUGAACGGUGAUCACCCGAGAUCCACCG GUCGCCACCAUG164nt-4xK1 UCAGAUCCGCUAGCGAUACACCGCAUCC 0.17 ± 44GGCGCGGAUUGGCCUGAACUGCCAGCUG 0.012 GCGCAGGUAGCAGAGCGGGUAAACUGGCUCGGAUUAGGGCCGCAAGAAAACUAUCC CGACCGCCUUACUGCCGCCUGUUUUGACCGCUGGGAUCUGCCAUUGAGAUCCGGGU GUGAACGGUGAUCACCCGAGAUCCGGGUGUGAACGGUGAUCACCCGAGAUCCGGGU GUGAACGGUGAUCACCCGAGAUCCGGGUGUGAACGGUGAUCACCCGAGAUCCACCG GUCGCCACCAUG

In Table 6, the initiation codon is shown in bold, and each underlinedportion corresponds to the Kl motif.

The translational efficiency of these sixteen mRNAs was measured in thesame manner as in Examples 1 and 2. The results are shown in FIGS. 7 and8. It is understood, as illustrated in graphs of FIGS. 7 and 8, that thetranslational efficiency of the EGFP is more decreased as the number ofKl motifs is larger and as the Kl motif is positioned closer to the 5′terminus of the mRNA. In other words, it was found that thetranslational efficiency can be rather precisely controlled by using twoparameters, the number of inserted Kl motifs and the insertion positionof the Kl motif. In FIG. 8, the translational efficiency of an mRNA intowhich one Kt motif was similarly inserted instead of the Kl motif isalso shown in the graph for comparison. In the sixteen mRNAs into whichthe Kl motifs were inserted, the translational regulatable range for thetarget protein was approximately 3% to approximately 75%.

Furthermore, an mRNA into which a Kink loop 2 motif obtained bymodifying the Kl motif was similarly inserted was designed, and aplasmid was constructed. The Kink loop 2 motif is hereinafter referredto as the Kl2 motif FIG. 5(B) illustrates the structure of the Kl2motif. In this experiment, two mRNAs were designed. In one of the mRNAs,one Kl2 motif was inserted at the 32th base from the 5′ terminus of themRNA. The other was designed to contain two Kl2 motifs with a distanceof the Kl motif positioned closer to the 5′ terminus from the 5′terminus set to 32nd base from the 5′ terminus. The details of the mRNAsare shown in Table 7 below. The open reading frame was set to encode theECFP gene. A vector was prepared for each of the mRNAs, transfected intoa cell in which the L7Ae or MS2CP was present, and the translationalefficiency was measured by the flow cytometry. As a result of threeexperiments, the average translational efficiency of the mRNAs intowhich one Kl2 motif was inserted was 0.84, whereas the averagetranslational efficiency of the mRNAs into which two Kl2 motifs wereinserted was 0.093.

TABLE 7 Trans- SEQ lational ID construct Sequences of the 5′ UTRefficiency NO K12 UCAGAUCCGCUAGCGCUACCGGACUCAG 0.84 ± 45AUCCGGACGUACGUGUGAACGGUGAUCA 0.0019 CGUACGCCGAGAUCCACCGGUCGCCACC AUG2xK12 UCAGAUCCGCUAGCGCUACCGGACUCAG 0.093 ± 46AUCCGGACGUACGUGUGAACGGUGAUCA 0.0064 CGUACGCCGAGAUCCGGACGUACGUGUGAACGGUGAUCACGUACGCCGAGAUCCAC CGGUCGCCACCAUG

In Table 7, an initiation codon is shown in bold, and each underlinedportion corresponds to the Kl2 motif.

In order to confirm whether the expression of a reporter gene wasregulated after transcription, the amount of a designed mRNA temporarilyexpressed in a cell was measured by real time quantitative PCR. In thisexperiment, four mRNAs in which one or four Kl motifs were inserted anda distance from the 5′ terminus of the Kl motif positioned closest tothe 5′ terminus was 18 bases or 164 bases were used for the measurement.The results are shown in FIG. 10. It was confirmed, based on a graph ofFIG. 10, that the relative transcription level of the mRNA is affectedby neither the structure of the 5′UTR nor the binding of a triggerprotein.

EXAMPLE 4

[Simultaneous Control of Two Different mRNAs in Single Cell]

Next, it was examined whether or not a single trigger protein of aneffector molecule can simultaneously and independently regulateexpression of multiple genes respectively having differently controlledcis-regulatory factors. FIG. 9 is a conceptual diagram of thisexperiment. A plasmid set composed of a first reporter plasmid and asecond reporter plasmid respectively encoding mRNAs that respectivelyhave RNA motifs specifically binding to the same trigger protein but aredifferent in the structure of the 5′UTR regulation structure wasdesigned. These plasmids were transfected into a cell in which thetrigger protein specifically binding to the RNA motif was present andinto a cell in which the trigger protein was absent. The first reporterplasmid was an mRNA encoding an EGFP and having one Kt motif or dKtmotif in the 5′ position (see the structure of FIG. 2(A)). The secondreporter plasmid was an mRNA encoding an ECFP (enhanced cyan fluorescentprotein) and having, in the 5′UTR, a Kl motif two-dimensionally arrangedas one used in Example 3. Respective sets of the first reporter plasmidand the second reporter plasmid were constructed so as to express mRNAshaving expression efficiency regulated respectively at three stages.Constructs with low expression efficiency were Kt-EGFP and18nt-3xKl-ECFP, constructs with high expression efficiency were dKt-EGFPand 120nt-1xKl-ECFP, and constructs with intermediate expressionefficiency were Sp-Kt-EGFP and 67nt-3xKl-ECFP. They are shown in Table 8below. These nine sets are respectively designated by using numbersshown in Table 8.

TABLE 8 Kt-EGFP Sp-Kt-EGFP dKt-EGFP 120nt-1xKl-ECFP (1) (4) (7)67nt-3xKl-ECFP (2) (5) (8) 18nt-3xKl-ECFP (3) (6) (9)

In the absence of the L7Ae protein functioning as the trigger proteinfor both the Kt motif and the Kl motif, the EGFP and the ECFP wereuniformly expressed in all the cells transfected with the nine sets andthe expression was not affected by the different structures of the5′UTRs. On the other hand, in the presence of the L7Ae protein, theexpression efficiency of the EGFP and the ECFP differs depending uponthe structures of the 5′UTRs of the respective mRNAs, and nine differentfluorescence profiles were obtained. The results are shown in FIG. 11.Respective numbers shown in FIG. 11 correspond to the sets numbered inTable 8 above. The output of each mRNA, namely, the amount of proteinproduced by the translation of each mRNA, is different from the outputsof the other mRNAs, and this means that the L7Ae protein cansimultaneously and independently regulate the translation of the twomRNAs different in the 5′UTR regulation structure and in the targetprotein gene.

EXAMPLE 5

[Approach Using Another RNP Motif]

Next, an experiment was carried out for confirming whether or not thistranslational repression can be similarly exhibited in an mRNA providedwith another RNP motif in the 5′UTR regulation structure. This exampleshows that similar effects can be attained also in a combination of theMS2 coat protein and an RNA motif specifically binding thereto and acombination of the Bacillus ribosomal protein S15 and an RNA motifspecifically binding thereto.

(a) MS2 Coat Protein

FIG. 12(A) illustrates a secondary structure of an MS2 stem-loop motif,that is, an RNA motif to which the MS2 coat protein specifically binds.The MS2 stem-loop motif is hereinafter referred to as the MS2SL motif.Four mRNAs each provided with the 5′UTR to which the MS2SL motif wasinserted were prepared. The number of the inserted MS2SL motifs wasaltered between one and two, and a distance of the MS2SL motifpositioned closest to the 5′ terminus from the 5′ terminus was alteredbetween 18 bases and 67 bases. The open reading frame and the structureof the 3′UTR were the same in all the four mRNAs. The details are shownin Table 9 below.

TABLE 9 Trans- lational SEQ ID construct Sequences of the 5′ UTRefficiency NO 18nt-1xMS2 UCAGAUCCGCUAGGAUCCGGUGAGGAUC 0.48 ± 47 SLACCCAUCGAGAUCCACCGGUCGCCACCA 0.16 UG 18nt-2xMS2UCAGAUCCGCUAGGAUCCGGUGAGGAUC 0.18 ± 48 SL ACCCAUCGAGAUCCGGUGAGGAUCACCC0.042 AUCGAGAUCCACCGGUCGCCACCAUG 67nt-1xMS2 UCAGAUCCGCUAGCCCGACCGCCUUACU1.0 ± 49 SL GCCGCCUGUUUUGACCGCUGGGAUCUGC 0.35CAUUGAGAUCCGGUGAGGAUCACCCAUC GAGAUCCACCGGUCGCCACCAUG 67nt-2xMS2UCAGAUCCGCUAGCCCGACCGCCUUACU 0.67 ± 50 SL GCCGCCUGUUUUGACCGCUGGGAUCUGC0.22 CAUUGAGAUCCGGUGAGGAUCACCCAUC GAGAUCCGGUGAGGAUCACCCAUCGAGAUCCACCGGUCGCCACCAUG

In Table 9, an initiation codon is shown in bold, and each underlinedportion corresponds to the MS2SL motif.

These four mRNAs were introduced into HeLa cells, so as to examine thetranslational efficiencies. FIG. 13 illustrates the result obtained byexpressing the mRNAs containing the MS2SL motifs in the presence of theMS2 coat protein. It is understood, also in this case, in the samemanner as in using the combination of the L7Ae protein and the Kt motifor the Kl motif, that the translational efficiency can be more decreasedas the number of motifs is larger and as the insertion position of themotif is closer to the 5′ terminus of the mRNA.

(b) Bacillus Ribosomal Protein S15

FIG. 12(B) illustrates a secondary structure of an Fr15 motif, that is,an RNA motif to which the Bacillus ribosomal protein S15 binds. FourmRNAs, each provided with the 5′UTR to which the Fr15 motif wasinserted, were prepared. The number of the inserted Fr15 motifs wasaltered between one and two, and a distance of the Fr15 motif positionedclosest to the 5′ terminus from the 5′ terminus was altered between 18bases and 67 bases. The open reading frame and the structure of the3′UTR were the same in all the four mRNAs. The details are shown inTable 10 below.

TABLE 10 Trans- lational SEQ ID construct Sequences of the 5′ UTRefficiency NO 18nt-1xFr15 UCAGAUCCGCUAGGAUCCUCGGUCGAAA 0.31 ± 51GACUUGAGGGCAGGAGAGGACUUCGGUC 0.032 UGGCCUGCACCUGACGAGAUCCACCGGUCGCCACCAUG 18nt-2xFr15 UCAGAUCCGCUAGGAUCCUCGGUCGAAA 0.16 ± 52GACUUGAGGGCAGGAGAGGACUUCGGUC 0.0082 UGGCCUGCACCUGACGAGAUCCUCGGUCGAAAGACUUGAGGGCAGGAGAGGACUUC GGUCUGGCCUGCACCUGACGAGAUCCAC CGGUCGCCACCAUG67nt-1xFr15 UCAGAUCCGCUAGCCCGACCGCCUUACU 0.45 ± 53GCCGCCUGUUUUGACCGCUGGGAUCUGC 0.10 CAUUGAGAUCCUCGGUCGAAAGACUUGAGGGCAGGAGAGGACUUCGGUCUGGCCUG CACCUGACGAGAUCCACCGGUCGCCACC AUG67nt-2xFr15 UCAGAUCCGCUAGCCCGACCGCCUUACU 0.22 ± 54GCCGCCUGUUUUGACCGCUGGGAUCUGC 0.030 CAUUGAGAUCCUCGGUCGAAAGACUUGAGGGCAGGAGAGGACUUCGGUCUGGCCUG CACCUGACGAGAUCCUCGGUCGAAAGACUUGAGGGCAGGAGAGGACUUCGGUCUGG CCUGCACCUGACGAGAUCCACCGGUCGC CACCAUG

In Table 10, an initiation codon is shown in bold, and each underlinedportion corresponds to the Fr15 motif.

In the same manner as described in the item (a), these four mRNAs wereintroduced into HeLa cells, so as to examine the translationalefficiencies. FIG. 14 illustrates the result obtained by expressing themRNAs containing the Fr15 motifs in the presence of the Bacillusribosomal protein S15. It is understood, in the same manner as describedin the item (a), that the translational efficiency can be more decreasedas the number of Fr15 motifs is larger and as the insertion position ofthe Fr15 motif is closer to the 5′ terminus of the mRNA.

It is understood from the results of Example 5 that the translationalefficiency can be quantitatively regulated not only in using the Ktmotif, that is, the RNA binding motif of the L7Ae protein, and itsvariants, the Kl motif and the Kl2 motif, but also in using acombination of another RNA-protein complex motif-derived RNA motif and aprotein specifically binding thereto.

EXAMPLE 6

[Preparation of mRNA Having Inverter ON Switch Cassette]

As illustrated in FIGS. 17(A) and 17(B), a cassette composed of a baitORF (hRluc gene in this case), β globin intron and IRES was insertedbetween an RNA motif of Kt-EGFP (32nt-Kt) and an initiation codon of agene to be translated by the method described in the item (1) below (asan OFF switch). A cassette in which a stop codon (a prematuretermination codon (PTC)) was inserted at the 457th base and 466th base(tandem PTCs) from the initiation codon of the bait ORF was alsoprepared (as an ON switch cassette: SEQ ID NO: 55). When the ON switchcassette is inserted into an mRNA, the bait ORF is not translated but agene following the IRES sequence is translated by the function of thetrigger protein. Accordingly, it is conceivable that a desired protein(output) can be made to be translated in the presence of the triggerprotein (input). On the other hand, it is conceivable that in theabsence of the trigger protein (input), although the translation of thebait ORF proceeds, since the stop codon is positioned more than 500 bpupstream of the intron, the RNA decay is caused by the nonsensemutation-dependent mRNA decay mechanism (NMD), and hence the desiredprotein (output) cannot be translated because of the decay of the mRNA.Accordingly, a plasmid expressing an mRNA containing the ON switch andthe Kt motif or the dKt motif and a plasmid expressing an mRNA encodingL7Ae or MS2 were simultaneously introduced into a cell in a ratio of1:0.2. Thus, merely when the Kt motif and the L7Ae were simultaneouslyexpressed, the EGFP was expressed (FIG. 18(A)). Correspondingly to thisresult, the amount of mRNA measured 24 hours after introducing theplasmid by the PCR was larger when the Kt motif and the L7Ae weresimultaneously expressed (FIG. 18(B)). Flow cytometry analysis revealedthat cells transfected with the cognate pair of plasmids (L7Ae/ON-Kt)showed 5- to 7-fold higher levels of EGFP fluorescence in average thanthose with non-cognate pairs (FIG. 18(E)). These results indicate thatthe insertion of the ON switch cassette (cis-acting module) into themRNA effectively inverted the OFF switch to the ON switch.

The amount of switch mRNA present 24 hours after transfection wasdetermined via quantitative RT PCR analysis (FIG. 18(F)). As expected,the amount of ON switch mRNA in cells transfected with the cognate pairs(L7Ae/ON-Kt) was 1.7-fold higher than in cells with non-cognate pairs,showing that the inserted module increased the steady state levels ofthe switch mRNA in the cells.

To further investigate the molecular mechanism of the designed module, adefective module was constructed by removing the tandem PTCs, resultingin 43 nucleotide (nt) in length between the stop codon of the bait ORFand the splice site of the intron (referred to as ONn; FIG. 20(A)). ONnwas inserted to the parental OFF switch and it was found that removal ofPTCs increased EGFP production even in the absence of L7Ae, andtherefore disrupted the ability of the inverter module (FIG. 20(A)).This suggests that basal repression of the ON switch cassette depends onPTCs.

Next, NMD regulatory protein factors (SMG1, UPF1 and UPF2) were knockeddown by using short interference RNAs (siRNAs) (FIG. 20(B)). Two daysafter the transfection of siRNAs, the same sets of plasmids weretransfected and the behavior of the inverted switches was assessed.Knockdown of these factors increased EGFP expression and diminishedfurther up-regulation of EGFP expression in the absence and presence ofL7Ae, respectively (FIG. 20(C)), indicating that inversion of the switchdepends on these factors.

In addition, some construct of ON switch cassettes were produced, inwhich distance between PTCs and the spliced site of the module wasshortened (FIG. 20(C)). Shortening the distance to 320 nt was sufficientfor the module to invert the switch. However distances shorter thanthis, such as 160nt proved insufficient in keeping with previousevidence that the shorter distances were less effective for triggeringNMD. Taken together, these results indicate that the function of thisinverter module depends on the mechanism of NMD.

Subsequently, in order to confirm whether or not a similar effect can beattained by using another trigger protein, expression of the EGFP waschecked by combining the Kt or Fri 5 motif and the OFF switch or the Ktor Fri 5 motif and the ON switch with the various ratio of L7Ae or theS15 (input plasmid) to ON or OFF switch (output plasmid) (FIG. 19(A)).The observed correlation between the behavior of the S15 system in theparental OFF and inverted ON switches was similar to that of the L7Aesystem (FIG. 19(A); ON-Fr15 & OFF-Fr15). In addition, western blotanalyses were performed to determine the input protein levels under theexperimental conditions (FIGS. 19(C) and 19(D)). It was confirmed thatexpression level of L7Ae or S15 increases depending on the amounts ofthe input plasmid (0.2-5 fold to the output plasmid) and is notsaturated under our experimental conditions.

Furthermore, similar experiments were carried out by using two variantsof the Kt motif, that is, L7Ae (Kd value (dissociation constant) of 1.6nM), K37A variant (L7K: Kd value of 15 nM) and K78A double variant(L7KK: Kd value of 680 nM). Thus, it was observed that as thedissociation constant was greater, the expression of the EGFP wasdecreased in using the ON switch and the expression was increased inusing the OFF switch (FIG. 19(B) and FIG. 22). These data indicate thatthe switch-inverting module can universally derive ON switches fromparental OFF switches. The ON and OFF switches show similar efficienciesin responding to the same input in relation to the amount of the inputmolecule and the affinity of the interaction between the input and thesensory RNA motif.

Ideally, the response of the inverted switch to the input signal shouldbe exactly the opposite of the response of the parental switch. Thedynamic range between the basal (repressed) and fully released state ofthe inverted ON-Fr15 switch was similar to that between the basal(released) and fully repressed state of the parental OFF-Fr15 switch(FIG. 19(A)). In contrast, the absolute output values corresponding tothe repressed and released states of the two switches were differentunder the same conditions. If the absolute values after the conversionneed to be adjusted for some applications, the mRNA levels in a cell canbe optimized by altering the strength of the promoter or the efficiencyof plasmid uptake; this procedure will likely compensate for thedifference by vertically shifting the curves shown in FIG. 19(A). Infact, dilution of the output plasmids altered the absolute values ofEGFP expression but maintained similar dynamic range before and afterthe inversion, suggesting that NMD component is not saturated under theExample conditions (FIG. 23(A)). It also revealed that this module iseffective under the control of alternative promoters such as RSVpromoter or EF1α promoter in addition to CMV promoter (FIG. 23(B)).These promoters altered the level of EGFP expression while maintaining asimilar fold change before and after the inversion as in the case ofplasmid dilution, indicating that output protein level from an invertedswitch can be tuned by employing different promoters and/or thedifferent concentration of the plasmids.

The minimum and maximum limits of the output expression determine theusable range of the inverted switch. In the case of the L7Ae-responsiveswitches described above, the corresponding usable range was narrowedafter the conversion of OFF-Kt into ON-Kt (FIG. 19(A)). The response ofON-Kt reached its maximal limit at an input protein level that onlysuppressed OFF-Kt to half of its maximal level (corresponding to morethan a 10-fold difference). The IRES-driven synthesis of the outputprotein is blocked after the IRES is degraded as a consequence of NMD.Therefore, very efficiently coupling NMD to IRES inactivation couldreduce the minimum output level of the system. Enhancing the activity ofthe IRES could also improve the performance of the module by increasingthe output level because IRES-driven protein synthesis is generally lesseffective than cap-dependent translation.

It was investigated whether our system could control a cellularphenotype via apoptosis pathways (FIG. 24(A)). It was already shown thatthe OFF switch could repress translation of anti-apoptotic Bcl-xL toinduce apoptosis (Saito, H., et al. Nat Commun 2, 160 (2011)). Likewise,an apoptosis-controllable switch was designed, in which the output EGFPprotein was replaced with pro-apoptotic Bim-EL to express Bim-EL in theinput-protein, L7Ae, dependent manner (FIG. 24(A)). After thetransfection of the corresponding plasmids, the number of AnnexinV-positive apoptotic cells was evaluated by using a flow cytometer. Asexpected, Annexin V-positive cells were increased specifically when thecognate pair of the plasmids was injected (L7Ae and ON-Kt-B, FIG.24(B)).

Simultaneous regulation of two independent mRNAs by the OFF switch andthe ON switch was performed (FIG. 25(A)). The behavior of the invertedswitches (ON-Kt or ON-dKt) and their modified parental switches (OFF-Ktor OFF-dKt) was analyzed at the same time (FIG. 25(A)). As expected,both ON and OFF switches containing Kt were specifically up- anddown-regulated EGFP and ECFP as an output, respectively, in the presenceof L7Ae. Notably, each OFF or ON switch incorporated into the same celldid not affect the function of its counterpart. Furthermore, analternative ON switch utilizing another input protein, S15, was employedand it was confirmed that the OFF (OFF-Kt or OFF-dKt) and ON (ON-Fr15 orON-dFr15) switches specifically respond to the corresponding inputs,L7Ae and S15, respectively (FIG. 25(B)).

Discussion

Signal inversion is one of the most fundamental processes in a circuit.Similar to electrical engineering, complicated biological circuitsutilize many signal inversions (Stapleton, J. A. et al. ACS Synth. Biol.1, 83-88 (2011), Xie, Z., Wroblewska, L., Prochazka, L., Weiss, R. &Benenson, Y. Science 333, 1307-1311 (2011), and Wang, B., Kitney, Joly,N. & Buck, M. Nat Commun 2, 508 (2011)). In many instances, syntheticbiologists have employed trans-acting effector and sensor pairs asinverter modules. This approach requires at least one unique regulatorypair for every inversion, and performing the inversion in a cellrequires a highly independent pair to avoid crosstalk between signalingcircuits, which would likely represent a major problem. To avoid thispotential pitfall, recent efforts have been made to generate numerousorthogonal regulatory pairs (Mutalik, V. K., Qi, L., Guimaraes, J. C.,Lucks, J. B. & Arkin, A. P. Nat. Chem. Biol. 8, 447-454 (2012)).Unfortunately, while many orthogonal pairs could potentially bedeveloped to generate new sets of trans-acting effectors and sensors,the number of such sets is finite. In contrast, the ON switches producedby the cis-acting module allow the input molecules to directly determinethe output protein levels in the absence of additional factors.Moreover, the cis-acting module is advantageous when compared withtrans-acting modules because it will likely enable the inversion ofmultiple signals with similar efficiency. The dynamic range of eachinverted switch will differ and will be determined by the nature of thecorresponding module in the case of trans-acting modules.

This invention succeeded in replacing the original β-globin intron (476nt) with the shorter chimeric intron (133 nt) without sacrificing theefficiency of the inverter module (FIG. 21(B)). Moreover, the modulewith a shortened distance between PTC and the splice site of the intron(320nt) maintained the efficiency (FIG. 21(A)). These results indicatethat more compact modules could be designed and constructed by usingthese elements. Furthermore, a module containing another bait ORF (thepart of EGFP gene) functioned as an inverter (FIGS. 25(A) and 25(B)).Thus, a bait ORF is likely to be replaced with desired protein-codingsequences.

This module could be employed to develop new ON switches from availabletranslational OFF switches in which the 5′-UTRs of the mRNAs respond toa variety of input molecules, including small molecules, RNA, andproteins, in eukaryotic cells (Saito, H. et al. Nat. Chem. Biol. 6,71-78 (2010), Saito, H., Fujita, Y., Kashida, S., Hayashi, K. & Inoue,T. Nat Commun 2, 160 (2011), Werstuck, G. & Green, M. R. Science 282,296-298 (1998), Hanson, S., Berthelot, K., Fink, B., McCarthy, J. E. &Suess, B. Mol. Microbiol. 49, 1627-1637 (2003), and Paraskeva, E.,Atzberger, A. & Hentze, M. W. Proc. Natl. Acad. Sci. U.S.A. 95, 951-956(1998)). In addition, it should also be possible for this module toinvert ON switches (Schlatter, S. & Fussenegger, M. Biotechnol. Bioeng.81, 1-12 (2003)) to OFF switches, because the IRES-driven production ofan output protein from the inverted switches is inversely related to theactivity of the cap-dependent translation of the bait ORF thatcorresponds to the output of the parental switches.

Finally, the method that we have shown in the present study enables thedetection of proteins in the cytoplasm while another synthetic RNAdevice has been reported that can detect nuclear protein expression tocontrol genetic circuits by employing alternative splicing (Culler, S.J., Hoff, K. G. & Smolke, C. D. Science 330, 1251-1255 (2010)). Thus,the two types of switches are now available that work in either thenucleus or cytoplasm.

The specific methods for preparing mRNAs and plasmid vectors expressingthe trigger proteins and for introducing them into a cell, and variousmeasurement methods commonly employed in Examples 1 to 6 will bedescribed in the following.

(1) Construction of Reporter Plasmid

In these examples, the pKt-EGFP and pdKt-EGFP used as a reporter plasmidexpressing the EGFP were prepared respectively correspondingly to plboxC/D-EGFP and pl boxC/D mutEGFP described by Gossen M, Bujard H (1992)Proc Natl Acad Sci USA 89:5547-5551. With the region encoding the EGFPreplaced by ECFP, pKt-ECFP and pdKt-ECFP were similarly prepared. Thesequences of the 5′UTRs of the pKt-EGFP and the pKt-ECFP accord withthat of the 32nt-Kt shown in Table 5 above.

The spacer sequences were amplified by the PCR from LacZ. Primer sets of(5′-CCCGGGATCCGATCCCGTCGTTTTACAAC-3′ (SEQ ID NO:56)/5′-AGATCTACCGGTCAGGCTGCGCAAC-3′ (SEQ ID NO: 57) and5′-GGATCCGCTAGCGATACACCGCATC-3′ (SEQ ID NO:58)/5′-ACTAGTAGATCTCAATGGCAGATCCCAG-3′ (SEQ ID NO: 59) were used. Aprimer set is herein mentioned as a forward primer and a reverse primerin this order. The spacer sequences were digested and ligated betweenthe BamHI-AgeI sites and the NheI-BglII sites of pKt-EGFP so as toprepare plasmids pKt-Sp-EGFP and pSp-Kt-EGFP, respectively. Similarly,pdKt-Sp-EGFP and pSp-dKt-EGFP were prepared from pdKt-EGFP.

The pKt-ECFP and pdKt-ECFP were digested with NheI and BamHI, blunted bya Klenow fragment (manufactured by Takara Bio Inc.) and self-ligated toprepare the shortest 5′UTR spacer (18 nt). The longest spacer sequence(320 nt) was obtained by concatenation of the two spacer fragmentsdescribed above and inserted between the NheI-BglII sites of pKt-ECFPand pdKt-ECFP. The other spacers were obtained by amplifying appropriateprimer sets inserted into the 5′UTRs of the reporter plasmids in asimilar manner. The sequences of all the used 5′UTRs are shown in Table5 above.

Pairs of oligonucleotides Kl were5′-CATGGGATCCGGGTGTGAACGGTGATCACCCGA-3′ (SEQ ID NO:60)/5′-GATCTCGGGTGATCACCGTTCACACCCGGATCC-3′ (SEQ ID NO: 61). Pairs ofoligonucleotides Kl2 were5′-CATGGGATCCGGACGTACGTGTGAACGGTGATCACGTACGCCGA-3′ (SEQ ID NO:62)/5′-GATCTCGGCGTACGTGATCACCGTTCACACGTACGTCCGGATCC-3′ (SEQ ID NO: 63).Pairs of oligonucleotides MS2SL were 5′-CATGGGATCCGGTGAGGATCACCCATCGA-3′(SEQ ID NO: 64)/5′-GATCTCGTTGGGTGTTCCTCTCCGGATCC-3′ (SEQ ID NO: 65).These nucleotide pairs were annealed and cloned into a cloning vector.The Fr15 was amplified by the PCR from DNA templates(5′-GGGATGTCAGGTGCAGGCCAGACCGAAGTCCTCTCCTGCCCTCAAGTCTTTCGACCATCCCTATAGTGAGTCGTATTAGC-3′ (SEQ ID NO: 66)) by using primer sets(5′-GCTAATCCATGGGATCCTCGGTCGAAAGACTTGAGGGC-3′ (SEQ ID NO:67)/5′-CCCAGATCTCGTCAGGTGCAGGCCAGAC-3′ (SEQ ID NO: 68)). The Fr15 wasthen digested by NcoI and BglII and cloned into the cloning vectorsimilarly. Each RNA motif was concatenated by using BamHI at the 5′terminus and BglII at the 3′ terminus of the cloning vector. Then,single or multiple RNA motifs were extracted from the cloning vector bydigestion with BamHI and BglII and inserted between the BglII-BamHIsites of the vectors that contain the Kt motif at the 67th, 120th and164th nucleotides from the 5′ terminus. The same fragments of RNA motifswere also inserted into the BamHI site of pKt-ECFP and placed at the18th nucleotide from the 5′ terminus by blunting and self-ligation.

An ON switch cassette was constructed from Renilla luciferase (hRluc)with nonsense mutation, β globin intron and IRES2. Briefly, it wasprepared as follows: pLP1 (Invitrogen Corporation) was digested withBamH1 and BglII, and β globin intron was extracted and inserted into theBamH1 site of pIRES2-EGFP (Clontech Laboratories, Inc.). The thusobtained plasmid was digested with BamH1, blunted by a Klenow fragmentand then self-ligated to remove the BamH1 site (psBIntIRES2-EGFP). A5′UTR digested from pl boxC/D-EGFP was inserted into NheI-NcoI site ofpGL4.73, and W153 and W156 of Renilla luciferase were transformed into astop codon by the PCR by using a primer set(5′-GTGACCTGACATCGAGGAGGATA-3′ (SEQ ID NO:69)/5′-TCGTCTCAGGACTCGATCACGTCC-3′ (SEQ ID NO: 70)). Subsequently, the5′UTR and the variant Renilla luciferase were inserted into thepsBIntIRES2-EGFP by digestion with NheI-SmaI site, so as to prepare aplasmid having an ON switch cassette. A plasmid having an OFF switchcassette was similarly prepared by using Renilla luciferase notconverted into a stop codon.

(2) Preparation of Trigger Plasmid

A trigger plasmid expressing a protein specifically binding to a Ktmotif was prepared. The protein specifically binding to the Kt motif isfused to a One-STrEP-tag (IBA) at the N-terminus and binds to a myc-tagand His-tag at the C-terminus, and has IRES-driven DSRed Express underthe control of a CMV promoter.

pIRES2-DsRed-Express was digested with BamHI and NotI, and a fragmentcontaining the IRES2-driven DsRed-Express expression cassette was clonedinto the BamHI-NotI site of pcDNA5/FRT/TO (manufactured by Invitrogen).A HindIII-digested fragment from p4LambdaN22-3mEGFP-M9 was inserted intothe resulting expression vector. Then four-times repeated Lambda N22peptide was replaced by the RNA-binding proteins that were amplified bythe PCR and fused to the peptide tags. The open reading frames ofArchaeoglobus fulgidus L7Ae were amplified by using a primer set(5′-GAATCCATGGGATCCATGTACGTGAGATTTGAGGTTC-3′ (SEQ ID NO:71)/5′-CACCAGATCTCTTCTGAAGGCCTTTAATCTTCTC-3′ (SEQ ID NO: 72)). The openreading frames of bacteriophage MS2 coat protein were amplified by usinga primer set (5′-CACCATGGGATCCGCTTCTAACTTTACTCAGTTCGTTCTC-3′ (SEQ ID NO:73)/5′-TATGAGATCTGTAGATGCCGGAGTTGGC-3′ (SEQ ID NO: 74)). Furthermore,the open reading frames of Bacillus stearothermophilus S15 wereamplified by using a primer set (5′-GACACCATGGGATCCGCATTGACGCAAGAGCG-3′(SEQ ID NO: 75)/5′-TATGAGATCTTCGACGTAATCCAAGTTTCTCAAC-3′ (SEQ ID NO:76)). These primers were respectively derived from a plasmid pL7Ae, aplasmid MS2-EGFP and a plasmid newly synthesized based on 25: Scott L G,Williamson J R (2001) Interaction of the Bacillus stearothermophilusribosomal protein S15 with its 5′-translational operator mRNA. J MolBiol 314:413-422.

RSV promoter and EF1α promoter were amplified via PCR using the primersets, 5′-GAGGGGGATTAATGTAGTCTTATGCAATACTCTTGTAGTCTTGC-3′ (SEQ ID NO:85)/5′-GTTGTTGCTAGCTCGAGCTTGGAGGTGC-3′ (SEQ ID NO: 86)and5′-GAATTCATTAATGGCTCCGGTGCCCGTCAG-3′ (SEQ ID NO:87)/5′-AAGCTTGCTAGCTCACGACACCTGAAATGGAAGAAAAAAAC-3′ (SEQ ID NO: 88),from pLP2 (Invitrogen) and KW239_p5E-hEF1α(kindly provided by Dr. K.Woltjen), respectively. They were digested by AseI and NheI forinsertion into AseI-NheI site of pON-Kt/dKt to generate pR-ON-Kt/dKt andpE-ON-Kt/dKt, respectively.

Shorter modules (ON32, ON16 and ON8) were generated via PCR-baseddeletion method using a reverse primer (5′-TGATCAGGGCGATATCCTCCTCG-3′(SEQ ID NO: 89)) with specific forward primers(5′-GTCCAGATTGTCCGCAACTACAACG-3′ (SEQ ID NO: 90),5′-GCCAGGAGGACGCTCCAG-3′ (SEQ ID NO: 91), and5′-TAGAGTCGGGGCGGCCGGGATC-3′ (SEQ ID NO: 92), respectively). Then,AgeI-Bsp1407I fragments of the resulting plasmids were returned intopON-Kt/dKt. To replace the intron, a chimeric intron and the hRluc genecontaining PTC were amplified via PCR using a primer set:5′-CGCAAATGGGCGGTAGGCGTG-3′ (SEQ ID NO:93)/5′-CATGGTTGTGGCCATATTATCATCG-3′ (SEQ ID NO: 94), and5′-CGATGATAATATGGCCACAACCATGGCAAAGCAACCTTCTGATG-3′ (SEQ ID NO: 95)/5′-GCCCCGC AGAAGGTCTAGAATCAATGCATTCTCCACACCAG-3′ (SEQ ID NO: 96), frompRL-TK (Promega) and pON-Kt, respectively. PCR products wereconcatenated via PCR together with the PCR product of IRES, digested byEcoRV and HindIII, and inserted into EcoRV-HindIII site of pON-Kt/dKt.Construction of pON2-Kt and pON2-dKt was similar to that of pON-Kt andpON-dKt, except for a primer set used to generate PTC:5′-CCCACCCTCGTGACCAC-3′ (SEQ ID NO: 97)/5′-TCAGGGCACGGGCAG-3′ (SEQ IDNO: 98).

IRES and Bim-EL were amplified from pIRES2-EGFP and pBim (Saito, H.,Fujita, Y., Kashida, S., Hayashi, K. & Inoue, T. Nat Commun 2, 160(2011).) via PCR using a primer set: 5′-CGCAAATGGGCGGTAGGCGTG-3′ (SEQ IDNO: 99)/5′-CATGGTTGTGGCCATATTATCATCG-3′ (SEQ ID NO: 100)and5′-CGATGATAATATGGCCACAACCATGGCAAAGCAACCTTCTGATG-3′ (SEQ ID NO:101)/5′-GCCCCGCAGAAGGTCTAGAATCAATGCATTCTCCACACCAG-3′ (SEQ ID NO: 102),respectively. These fragments were concatenated by PCR again using aprimer set: 5′-CGCAAATGGGCGGTAGGCGTG-3′ (SEQ ID NO:103)/5′-AAGCTTGCGGCCGCCCCGCAGAAGGTCTAGA-3′ (SEQ ID NO: 104), digestedwith HindIII and NotI, and inserted into HindIII-NotI site of pON-Kt/dKtto generate pON-Kt/dKt-B, respectively.

(3) Cell Culture and Transfections

HeLa cells were cultured at 37° C. and 5% CO₂ in Dulbecco's modifiedEagle's medium (GIBCO, Carlsbad, Calif.) containing 10% fetal bovineserum (Nichirei Biosciences, Tokyo, Japan) and 1% antibiotic-antimycoticsolution (Sigma-Aldrich, St Louis, Mo.). In all, 5×10⁴ cells were seededin 24-well plates, and after 24 hours, 70 to 90% confluent cells weretransiently transfected with plasmids using 1 μl of Lipofectamine 2000(Invitrogen, Carlsbad, Calif.) following the manufacturer'sinstructions. In the double-transfection experiments (Examples 1 to 3and 5), 0.1 μg of a reporter plasmid and 0.5 μg of a trigger proteinplasmid were transfected into cells. In the triple-transfectionexperiment (Example 4), 0.1 μg each of two reporter plasmids and 0.3 μgof a trigger protein plasmid were used. Media were changed 4 hours afterthe transfection.

(4) Flow Cytometric Measurement

Twenty-four hours after the transfection, cells were washed with PBS andincubated in 100 μl of 0.25% Trypsin-EDTA (GIBCO) for 2 min. at 37° C.After addition of 100 μl of the medium, cells were passed through a 35μm strainer (BD Biosciences, San Jose, Calif.) and then analyzed with aFACS Aria (BD Biosciences). A 408 nm semiconductor laser for excitationand a 450/40 nm filter were used to measure the fluorescence of ECFP. A488 nm semiconductor laser and 530/30 nm and 695/40 nm filters were usedto measure the fluorescence of EGFP and DsRed-Express, respectively.

(5) Flow Cytometric Analysis

Dead cells were gated out by using FSC (forward scatter property) andSCC (side scatter property). In this experiment, the translationalefficiency was defined as the ratio of an average of the EGFP or ECFPfluorescence intensity in the presence of RNA-binding protein (such asMC2CP), which is a negative control not binding to the RNA motif,divided by that in the presence of corresponding RNA-binding protein(such as L7Ae) from cells expressing a freely chosen 1000±100 a.u. ofDsRed. In the triple-transfection, the level of fluorescence wascompensated on the basis of cells respectively expressing the EGFP, theECFP or the DsRed-Express alone. Untransfected cells were gated outbased on the fluorescence level of DsRed-Express (<100 a.u.). All theexperiments were repeated three times, and the average and the standarddeviation are presented.

(6) Isolation of Total RNA and cDNA Synthesis

Twenty-four hours after the transfection, cells were washed with chilledPBS. Total RNA was isolated using the RNAqueous-4PCR Kit (manufacturedby Ambion), following the manufacturer's instructions. In all, 350 μl ofcell suspension/binding solution was used, and RNA was eluted in 50 μlof Milli-Q water twice. Contaminating DNA was removed using the TURBODNA-free Kit (manufactured by Ambion), following the manufacturer'sinstructions. cDNA was synthesized from 200 ng of the extracted totalRNA using High-Capacity cDNA Reverse Transcription Kits (manufactured byApplied Biosystems). Resulting cDNA solutions were diluted 10-fold, anda 5 μl aliquot was subjected to quantitative PCR analysis.

(7) Quantitative PCR Analysis

The quantitative PCR analysis was carried out using LightCycler 480 SYBRGreen I Master and LightCycler 480 instruments (Roche, Basel,Switzerland). The reaction solutions contained primer sets(5′-GAAGCGCGATCACATGGT-3′ (SEQ ID NO: 77)/5′-CCATGCCGAGAGTGATCC-3′ (SEQID NO: 78)) or (5′-GGCTACCCGTGATATTGCTG-3′ (SEQ ID NO:79)/5′-GCGATACCGTAAAGCACGA-3′ (SEQ ID NO: 80)) at a final concentrationof 500 nM to measure the RNA levels of reporter ECFP orneomycin-resistant gene, respectively. A series of 10-fold dilutions ofthe reporter plasmid (50 fg to 5 ng) was used as a standard. Therelative amount of the reporter ECFP mRNA was determined as a ratio tothat of neomycin-resistance gene that was expressed from the sameplasmid as the reporter. All the experiments were repeated three times,and the average and standard deviation were presented.

(8) RNAi Knock-down

A total of 2.5×10⁴ cells were seeded in 24-well plates, and after 24hours, cells were transfected with 20 pmol of siRNA using 1 micro-1 ofStemFect RNA transfection kit(Stemgent, Cambridge, Mass.) according tomanufacturer's instruction. The medium was changed 4 hours aftertransfection. After 48 hours, a set of plasmids were transfected asdescribed above. Then, after 24 hours, the cells were subjected to flowcytometry analysis using BD Accuri. According to the previous studies28, the sequences of siRNA used in this experiment were as follows:5′-GUGUAUGUGCGCCAAAGUATT-3′ (SEQ ID NO: 105)/5′-UACUUUGGCGCACAUACACTT-3′(SMG1) (SEQ ID NO: 106), 5′-GAUGCAGUUCCGCUCCAUUTT-3′ (SEQ ID NO:107)/5′-AAUGGAGCGGAACUGCAUCTT-3′ (UPF1) (SEQ ID NO: 108),5′-CAACAGCCCUUCCAGAAUCTT-3′ (SEQ ID NO: 109)/5′-GAUUCUGGAAGGGCUGUUGTT-3′(UPF2) (SEQ ID NO: 110), and 5′-UUCUCCGAACGUGUCACGUTT-3′ (SEQ ID NO:111)/5′-ACGUGACACGUUCGGAGAATT-3′ (n.c., non-silencing negative control)(SEQ ID NO: 112).

(9) Western Blotting Analysis

Transfection experiments were performed in four times larger scale in6-well plates compared with that in 24-well plates. After 24 hours(plasmid transfection) or 48 hours (RNAi knock-down), transfected cellswere washed twice with 2 mL of PBS and extracted in 0.3 mL of RIPAbuffer. Concentration of total proteins was measured by BCA Proteinassay (Thermo, Rockford, Ill.). Samples (10 micro-g) were subjected toSDS-PAGE, transferred into a PVDF membrane using iBlot (Invitrogen)following manufacture's instruction, and probed with indicatedantibodies followed by HRP-conjugated secondary antibodies. SMG1 andRENT1 antibody were purchased from Bethyl laboratories (Montgomery,Tex.). UPF2 rabbit monoclonal antibody (D3B10) was purchased from CellSignaling laboratory (Danvers, Mass.). The blots were detected withImmobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica,Mass.) and ImageQuant LAS 4000 (GE Healthcare, Piscataway, N.J.).

(10) Apoptosis Assay

Alternative INPUT plasmids, which express EGFP instead of DsRed-Express,were used in this experiment. Twenty-four hours after transfection ofthe plasmids, the cells and medium were collected, stained with AnnexinV, Pacific Blue conjugates (Invitrogen) according to the manufactures'instruction, and analyzed using a FACSAria cell sorter. Untransfectedcells were gated out based on EGFP fluorescence. The average andstandard deviation from two independent experiments are presented.

The invention claimed is:
 1. A translational regulation method using anRNA-protein interaction motif, comprising the step of: introducing anmRNA having: a 5′ UTR regulation structure comprising: (1) a capstructure on the 5′ terminus, (2) one or more RNA motifs positioned onthe 3′ side of the cap structure, of an RNA-protein interaction motifderived-nucleotide sequence or a variant thereof, and (3) an ON switchcassette positioned on the 3′ side of the one or more RNA motifs andhaving a sequence comprising (a) a bait open reading frame (a bait ORF),(b) an intron comprising a human beta globin intron or a chimericintron, and (c) an internal ribosome entry site (IRES); and a nucleotidesequence encoding a target protein gene on the 3′ side of the 5′ UTRregulation structure, into a cell, and starting translation of thetarget protein by a protein specifically binding to the one or more RNAmotifs, wherein the bait ORF is a sequence comprising a stop codon inmore than 320 bases from the intron, wherein said ON switch cassette ispositioned on (a) 3′ side of the RNA motif if the 5′ UTR regulationstructure comprises one RNA motif structure; or (b) 3′ side of the RNAmotif positioned at 3′ end of the RNA motifs if the 5′ UTR regulationstructure comprises two or more RNA motifs; wherein the mRNA is anartificially prepared non-natural mRNA; wherein said human beta globinintron comprises a nucleotide sequence of Seq. ID No. 18; wherein saidchimeric intron comprises a nucleotide sequence of Seq. ID No.
 83. 2.The method according to claim 1, wherein the bait ORF is a sequencecomprising a stop codon inserted at the 457th base from the 5′ side ofRenilla luciferase; wherein said Renilla luciferase comprises anucleotide sequence of Seq. ID No. 17 or Seq. ID No.
 81. 3. The methodaccording to claim 1, wherein the intron is human β globin intron. 4.The method according to claim 1, wherein each of the RNA motifs is abinding sequence selected from the group consisting of a bindingsequence of L7Ae protein, a binding sequence of MS2 phage coat proteinand a binding sequence of Bacillus stearothermophilus ribosomal proteinS15.